Antibodies directed to monocyte chemo-attractant protein-1 (MCP-1) and uses thereof

ABSTRACT

Embodiments of the invention described herein relate to antibodies directed to the antigen monocyte chemo-attractant protein-1 (MCP-1) and uses of such antibodies. In particular, in accordance with some embodiments, there are provided fully human monoclonal antibodies directed to the antigen MCP-1. Nucelotide sequences encoding, and amino acid sequences comprising, heavy and light chain immunoglobulin molecules, particularly sequences corresponding to contiguous heavy and light chain sequences spanning the framework regions and/or complementarity determining regions (CDRs), specifically from FR1 through FR4 or CDR1 through CDR3, are provided. Hybridomas or other cell lines expressing such immunoglobulin molecules and monoclonal antibodies are also provided.

PRIORITY CLAIM

This application claims priority under 35 U.S.C. § 119(e) to U.S.Provisional Application No. 60/404,802, filed Aug. 19, 2002, which ishereby expressly incorporated by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

Embodiments of the invention described herein relate to antibodiesdirected to the antigen monocyte chemo-attractant protein-1 (MCP-1) anduses of such antibodies. In particular, in accordance with embodimentsof the invention, there are provided fully human monoclonal antibodiesdirected to the antigen MCP-1. Nucleotide sequences encoding, and aminoacid sequences comprising, heavy and light chain immunoglobulinmolecules, particularly sequences corresponding to contiguous heavy andlight chain sequences spanning the framework regions and/orcomplementarity determining regions (CDRs), specifically from FR1through FR4 or CDR1 through CDR3, are provided. The antibodies of theinvention find use as diagnostics and as treatments for diseasesassociated with the overproduction of MCP-1. Hybridomas or other celllines expressing such immunoglobulin molecules and monoclonal antibodiesare also provided.

2. Description of the Related Art

An increased production of angiogenic factors and decreased productionof angiogenesis inhibitors by cancer cells, vascular endothelial cellsand other stromal cell types are believed to induce tumor angiogenesis.Stroma, comprised of interstitial connective tissues, basal lamina,blood cells, blood vessels and fibroblastic cells, surround almost allsolid tumor cells. Interactions between the stroma and cancer cells playa critical role in the neovascularization of tumors. Further,macrophage, which are also stromal components, are important in tumorangiogenesis. (M. Ono et al., Cancer Chemother. Pharmacol. (1999)43(Suppl.): S69–S71.)

Macrophages are the major terminally differentiated cell type of themononuclear phagocyte system, and are also one of the key angiogeniceffector cells, producing a number of growth stimulators and inhibitors.A number of angiogenic cytokines are known to be produced bymacrophages, including monocyte chemo-attractant protein 1 (MCP-1).

MCP-1 is known to be chemotactic for T lymphocytes, basophils and NKcells. MCP-1 is one of the most potent macrophage recruiting molecules.Once recruited to sites of inflammation or tumors, macrophages cangenerate a number of angiogenic cytokines, thereby stimulatingpathologic angiogenesis. A number of studies have shown a relationshipbetween angiogenesis, macrophage recruitment, and prognosis in patientswith various kinds of tumors (G. Fantanini et al., Int. J. Cancer (1996)67:615; N. Weidner et al., J. Natl. Cancer Inst. (1992) 84:1875). Leeket al. have further demonstrated that focally increased macrophagenumbers are closely related to vascularization and prognosis in breastcancer patients (Cancer Res. (1996) 56:4625). R. Huang et al. (CancerRes. (2002) 62:2806–2812) have shown that Connexin 43 suppresses humanglioblastoma cell growth by down regulation of MCP-1, as discovered byusing protein array technology.

Goede et al. (Int. J. Cancer (1999) 82: 765–770) first demonstrated thatMCP-1 had an angiogenic potency which was equivalent to that of VEGFwhen tested in a rabbit corneal model. In their model, the angiogenicactivity induced by MCP-1 was associated with an intense recruitment ofmacrophages into the rabbit cornea. Salcedo et al. have reported thatMCP-1 induced chemotaxis of human endothelial cells at nanomolarconcentrations. This chemotactic response was inhibited by a polyclonalantibody to human MCP-1 (R. Salcedo et al., Blood (2000) 96(1):34–40).

MCP-1 is the predominant chemokine expressed in ovarian cancer (Negus,R. P. M. et al., J. Clin. Investig. (1995) 95: 2391–96; Sica, A. et al.,J. Immunology (2000) 164(2):733–8). MCP-1 is also elevated in a numberof other human cancers including bladder, breast, lung, andglioblastomas.

In addition, the importance of MCP-1 in inflammation has been shown in anumber of studies. For example, H. J. Anders et al., have demonstratedchemokine and chemokine receptor expression during initiation andresolution of immune complex glomerulonephritis (J. Am. Soc. Nephrol.(2001) 12: 919–2001). Segerer et al. (J. Am. Soc. Nephrol. (2000)11:2231–2242) also have studied the expression of MCP-1 and its receptorchemokine receptor 2 in human crescentic glomerulonephritis. J. A.Belperio et al. have shown a critical role for the chemokine MCP-1/CCR2in the pathogenesis of bronchiolitis obliterans syndrome (J. Clin.Investig. (2001) 108: 547–556). N. G. Frangogiannis et al. havedelineated the role of MCP-1 in the inflammatory response in myocardialinfarction (Cardiovacular Res. (2002) 53: 31–47). Gerard and Rollins(Nature Immunol. (2001) 2:108–115) and Reape and Groot (Atherosclerosis(1999) 147: 213–225) have discussed the role of MCP-1 in atherosclerosisand other diseases. Also, Schmidt and Stem (Arterioscler. Thromb. Vasc.Biol. (2001) 21:297–299) describe MCP-1 interactions in restenosis.

Human MCP-1, a 76-amino-acid CC chemokine with an N-terminalpyroglutamic acid, was originally purified from several sourcesincluding phytohemagglutinin-stimulated human lymphocytes (Yoshimura, T.et al., J. Immunol. (1989) 142:1956–62), a human glioma cell line(Yoshimura, T., et al., J. Exp. Med. (1989) 169:1449–59), and the humanmyelomonocytic cell line THP-1 (Matsushima, K., et al., (1989) J. Exp.Med. (1989) 169: 1485–90). MCP-1 was first described aslymphocyte-derived chemotactic factor (LDCF). Other names for theprotein are tumor-cell-derived chemotactic factor (TDCF), glioma-derivedmonocyte chemotactic factor (TDCF), glioma-derived monocyte chemotacticfactor (GDCF), smooth muscle cell-derived chemotactic factor (SMC-CF),monocyte chemotactic activating factor (MCAF) and CCL2. Molecularcloning of the cDNA encoding MCP-1 (Furutani, Y., et al., (1989)Biochem. Biophys. Res. Comm. (1989) 169:249–55; B. J. Rollins, et al.,Mol. Cell. Biol. (1989) 9:4687–95; Chang, H. C., et al., Int. Immunol.(1989) 1:388–97) revealed an open reading frame of 99 amino acids,including a signal peptide of 23 amino acids. The mouse homologue geneof MCP-1 was named JE (B. J. Rollins et al., 1989).

WO 200189565, published Nov. 29, 2001, discloses polyclonal antibodiesto human MCP-1 and describes the inhibition of tumor growth in a nudemouse model by the use of such polyclonal antibodies.

Embodiments of the invention described herein relate to fully humanmonoclonal antibodies to human MCP-1 that block MCP-1-induced chemotaxisof THP-1 cells, a cell line derived from a patient with acute monocyticleukemia. These cells are used as a surrogate for assessing themigration of normal human mononuclear cells in circulation. Mononuclearcell infiltration stimulated by MCP-1 plays a pathologic role in anumber of inflammatory conditions including rheumatoid arthritis,glomerulonephritis, atherosclerosis, transplant rejection, psoriasis,restenosis, and autoimmune diseases such as multiple sclerosis. Anantibody that blocks MCP-1 activity and prevents monocyte infiltrationwill find use as a treatment for these and other inflammatory diseases.

SUMMARY OF THE INVENTION

Embodiments of the invention described herein related to monoclonalantibodies that were found to bind MCP-1 and affect MCP-1 function.Other embodiments relate to human anti-MCP-1 antibodies and anti-MCP-1antibody preparations with desirable properties from a therapeuticperspective, including strong binding affinity for MCP-1, the ability toneutralize MCP-1 in vitro, and the ability to inhibit neovascularizationof solid tumors.

One embodiment of the invention is a fully human monoclonal antibodythat binds to MCP-1 and has a heavy chain amino acid sequence selectedfrom the group consisting of SEQ ID NOS: 2, 6, 10, 14, 18, 22, 26, 30,34, 38, 42, 46, 50, 54, 58, 62, 66, 70, 74, 78, 82, 86, 90, 94, 98, 102,106, 110, 114, 118, 122, 126, 130, 134, 138, 142 and 146. In oneembodiment, the antibody further comprises a light chain amino acidsequence selected from the group consisting of SEQ ID NOS: 4, 8, 12, 16,20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, 80, 84, 88,92, 96, 100, 104, 108, 112, 116, 120, 124, 128, 132, 136, 140, 144 and148.

Accordingly, one embodiment of the invention described herein providesisolated antibodies, or fragments of those antibodies, that bind toMCP-1. As known in the art, the antibodies can advantageously be, forexample, monoclonal, chimeric and/or human antibodies. Embodiments ofthe invention described herein also provide cells for producing theseantibodies.

Another embodiment of the invention is a fully human antibody that bindsto MCP-1 that comprises a heavy chain amino acid sequence having theCDRs comprising the sequences shown in FIGS. 7 and 10. It is noted thatCDR determinations can be readily accomplished by those of ordinaryskill in the art. In general, CDRs are presented in the inventiondescribed herein as defined by Kabat et al., in Sequences of Proteins ofImmunological Interest vols. 1–3 (Fifth Edition, NIH Publication91–3242, Bethesda Md. 1991).

Yet another embodiment of the invention is a fully human antibody thatbinds to MCP-1 and comprises a light chain amino acid sequence havingthe CDRs comprising the sequences shown in FIGS. 8 and 9.

A further embodiment of the invention is a fully human antibody thatbinds to MCP-1 and comprises a heavy chain amino acid sequence havingthe CDRs comprising the sequences shown in FIGS. 7 and 10 and a lightchain amino acid sequence having the CDRs comprising the sequences shownin FIGS. 8 and 9.

Another embodiment of the invention is a fully human antibody that bindsto other MCP-1 family members including, but not limited to, MCP-2,MCP-3 and MCP-4. A further embodiment of the invention is an antibodythat cross-competes for binding to MCP-1 with the fully human antibodiesof the invention.

It will be appreciated that embodiments of the invention are not limitedto any particular form of an antibody or method of generation orproduction. For example, the anti-MCP-1 antibody may be a full-lengthantibody (e.g., having an intact human Fc region) or an antibodyfragment (e.g., a Fab, Fab′ or F(ab′)₂). In addition, the antibody maybe manufactured from a hybridoma that secretes the antibody, or from arecombinantly produced cell that has been transformed or transfectedwith a gene or genes encoding the antibody.

Other embodiments of the invention include isolated nucleic acidmolecules encoding any of the antibodies described herein, vectorshaving an isolated nucleic acid molecules encoding any of such theanti-MCP-1 antibodies, a host cell transformed with any of such nucleicacid molecules. In addition, one embodiment of the invention is a methodof producing an anti-MCP-1 antibody by culturing host cells underconditions wherein a nucleic acid molecule is expressed to produce theantibody followed by recovering the antibody.

A further embodiment of the invention includes a method of producinghigh affinity antibodies to MCP-1 by immunizing a mammal with humanMCP-1 or a fragment thereof and one or more orthologous sequences orfragments thereof.

Embodiments of the invention described herein are based upon thegeneration and identification of isolated antibodies that bindspecifically to MCP-1. MCP-1 is expressed at elevated levels inneoplastic diseases, such as tumors, and other inflammatory diseases.Inhibition of the biological activity of MCP-1 can prevent furtherinfiltration of mononuclear cells into tissues.

Another embodiment of the invention includes a method of diagnosingdiseases or conditions in which an antibody prepared according to theinvention described herein is utilized to detect the level of MCP-1 in apatient sample. In one embodiment, the patient sample is blood or bloodserum. In further embodiments, methods for the identification of riskfactors, diagnosis of disease, and staging of disease is presented whichinvolves the identification of the overexpression of MCP-1 usinganti-MCP-1 antibodies.

In another embodiment, the invention includes a method for diagnosing acondition associated with the expression of MCP-1 in a cell, comprisingcontacting the cell with an anti-MCP-1 antibody, and detecting thepresence of MCP-1. Preferred conditions include, but are not limited to,neoplastic diseases including, without limitation, tumors, cancers, suchas breast, ovarian, stomach, endometrial, salivary gland, lung, kidney,colon, colorectal, thyroid, pancreatic, prostate and bladder cancer, aswell as other inflammatory conditions, including, but not limited to,rheumatoid arthritis, glomerulonephritis, atherosclerosis, psoriasis,organ transplants, restenosis and autoimmune diseases.

In another embodiment, the invention includes an assay kit for thedetection of MCP-1 and MCP-1 family members in mammalian tissues orcells to screen for neoplastic diseases or inflammatory conditions,comprising an antibody that binds to MCP-1 and a means for indicatingthe reaction of the antibody with the antigen, if present. Preferablythe antibody is a monoclonal antibody. In one embodiment, the antibodythat binds MCP-1 is labeled. In another embodiment the antibody is anunlabeled first antibody and the means for indicating the reactioncomprises a labeled second antibody that is an anti-immunoglobulin.Preferably the antibody is labeled with a marker selected from the groupconsisting of a fluorochrome, an enzyme, a Radionuclide and a radiopaquematerial.

Other embodiments of the invention include pharmaceutical compositionscomprising an effective amount of the antibody of the invention inadmixture with a pharmaceutically acceptable carrier or diluent. In yetother embodiments, the anti-MCP-1 antibody or fragment thereof isconjugated to a therapeutic agent. The therapeutic agent can be a toxinor a radioisotope. Preferably, such antibodies can be used for thetreatment of diseases, such as, for example, tumors, including, withoutlimitation, cancers, such as breast, ovarian, stomach, endometrial,salivary gland, lung, kidney, colon, colorectal, thyroid, pancreatic,prostate and bladder cancer, as well as other inflammatory conditions,including, but not limited to, rheumatoid arthritis, glomerulonephritis,atherosclerosis, psoriasis, organ transplants, restenosis and autoimmunediseases.

Yet another embodiment of the invention provides a method for treatingdiseases or conditions associated with the expression of MCP-1 in apatient, comprising administering to the patient an effective amount ofan anti-MCP-1 antibody. The method can be performed in vivo. The patientis a mammalian patient, preferably a human patient. In a preferredembodiment, the method concerns the treatment of tumors, including,without limitation, cancers, such as breast, ovarian, stomach,endometrial, salivary gland, lung, kidney, colon, colorectal, thyroid,pancreatic, prostate and bladder cancer. In another embodiment, themethod concerns the treatment of inflammatory conditions, including, butnot limited to, rheumatoid arthritis, glomerulonephritis,atherosclerosis, psoriasis, organ transplants, restenosis and autoimmunediseases. Additional embodiments include methods for the treatment ofdiseases and conditions associated with the expression of MCP-1, whichcan include identifying a mammal in need of treatment for overexpressionof MCP-1 and administering to the mammal, a therapeutically effectivedose of anti-MCP-1 antibodies.

In another embodiment, the invention provides an article of manufacturecomprising a container, comprising a composition containing ananti-MCP-1 antibody, and a package insert or label indicating that thecomposition can be used to treat neoplastic and inflammatory diseasescharacterized by the overexpression of MCP-1. Preferably a mammal, andmore preferably, a human receives the anti-MCP-1 antibody. In apreferred embodiment, tumors, including, without limitation, cancers,such as breast, ovarian, stomach, endometrial, salivary gland, lung,glioblastomas, kidney, colon, colorectal, thyroid, pancreatic, prostateand bladder cancer, as well as other inflammatory conditions, including,but not limited to, rheumatoid arthritis, glomerulonephritis,atherosclerosis, psoriasis, organ transplants, restenosis and autoimmunediseases such as multiple sclerosis are treated.

In some embodiments, the anti-MCP-1 antibody is administered, followedby a clearing agent to remove circulating antibody from the blood.

In some embodiments, anti-MCP-1 antibodies can be modified to enhancetheir capability of fixing complement and participating incomplement-dependent cytotoxicity (CDC). In one embodiment, theanti-MCP-1 antibody can be modified, such as by an amino acidsubstitution, to alter antibody clearance. For example, certain aminoacid substitutions may accelerate clearance of the antibody from thebody. Alternatively, the amino acid substitutions may slow the clearanceof antibody from the body. In other embodiments, the anti-MCP-1 antibodycan be altered such that it is eliminated less rapidly from the body.

Yet another embodiment is the use of an anti-MCP-1 antibody in thepreparation of a medicament for the treatment of diseases such asneoplastic diseases and inflammatory conditions. In one embodiment, theneoplatic diseases include tumors and cancers, such as breast, ovarian,stomach, endometrial, salivary gland, lung, kidney, colon, colorectal,thyroid, pancreatic, prostate and bladder cancer. In an alternativeembodiment, the inflammatory condition includes, but is not limited to,rheumatoid arthritis, glomerulonephritis, atherosclerosis, psoriasis,organ transplants, restenosis and autoimmune diseases.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows results of THP-1 monocyte migration studies in response toMCP-1, MCP-2, MCP-3 and MCP-4.

FIG. 2 shows inhibition by antibody 3.11.2 in a dose-dependent manner ofthe migration ability of THP-1 cells in response to MCP-2.

FIG. 3 shows inhibition by antibody 3.11.2 in a dose-dependent manner ofthe migration ability of THP-1 cells in response to MCP-3.

FIG. 4 shows the effect of anti-MCP-1 antibody 1.7.3 on pancreatic tumorPanc-1 growth.

FIG. 5 shows a 3-dimensional scatter plot of calcium flux, chemotaxisand affinity data for the MCP-1 antibodies.

FIG. 6 shows another orientation of a 3-dimensional scatter plot ofcalcium flux, chemotaxis and affinity data for the MCP-1 antibodies.

FIG. 7A shows a Clustal W comparison of anti-MCP-1 sequences usingVH1-24, indicating the CDR1, CDR2, and CDR3 regions, and the associateddendrogram (FIG. 7B).

FIG. 8A shows a Clustal W comparison of anti-MCP-1 sequences usingVK-B3, indicating the CDR1, CDR2, and CDR3 regions, and the associateddendrogram (FIG. 8B).

FIG. 9A shows a Clustal W comparison of anti-MCP-1 sequences usingVK-08, indicating the CDR1, CDR2, and CDR3 regions, and the associateddendrogram (FIG. 9B).

FIG. 10A shows a Clustal W comparison of anti-MCP-1 sequences usingVH6-1, indicating the CDR1, CDR2, and CDR3 regions, and the associateddendrogram (FIG. 10B).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

Embodiments of the invention described herein relate to monoclonalantibodies that bind to MCP-1. In some embodiments, the antibodies bindto MCP-1 and affect MCP-1 function. Other embodiments provide fullyhuman anti-MCP-1 antibodies and anti-MCP-1 antibody preparations withdesirable properties from a therapeutic perspective, including strongbinding affinity for MCP-1, the ability to neutralize MCP-1 in vitro,and the ability to inhibit the growth and neovascularization of solidtumors in vivo.

Accordingly, embodiments of the invention provide isolated antibodies,or fragments of those antibodies, that bind to MCP-1. As known in theart, the antibodies can advantageously be, e.g., monoclonal, chimericand/or human antibodies. Embodiments of the invention also provide cellsfor producing these antibodies.

In some embodiments, the antibodies described herein possess therapeuticutilities. An anti-MCP-1 antibody can potentially block or limit theextent of tumor neovascularization and tumor growth. Many cancer cellsincluding those from glioblastomas and renal cancers express thereceptor for MCP-1, CCR2. The co-expression of ligand and receptor inthe same tumor cell suggests that MCP-1 may regulate an autocrine growthloop in cancer cells that express both components. Huang et al. (CancerRes. (2002) 62:2806–2812) have recently reported that MCP-1 can directlyinfluence the growth and survival of tumor cells that express the CCR2receptor for MCP-1. Thus, in addition to its effects on angiogenesis,MCP-1 may also directly regulate tumor cell growth, migration andinvasion.

In addition, embodiments of the invention provide for using theseantibodies as a diagnostic or treatment for disease. For example,embodiments of the invention provide methods and antibodies forinhibition expression of MCP-1 associated with tumors and inflammatoryconditions. Preferably, the antibodies are used to treat cancers, suchas breast, ovarian, stomach, endometrial, salivary gland, lung, kidney,colon, colorectal, thyroid, pancreatic, prostate and bladder cancer, aswell as other inflammatory conditions, including, but not limited to,rheumatoid arthritis, glomerulonephritis, atherosclerosis, psoriasis,organ transplants, restenosis and autoimmune diseases. In associationwith such treatment, articles of manufacture comprising antibodies ofthe invention described herein are provided. Additionally, an assay kitcomprising antibodies in accordance with the invention described hereinis provided to screen for tumors and inflammatory conditions.

Additionally, the nucleic acids described herein, and fragments andvariants thereof, may be used, by way of nonlimiting example, (a) todirect the biosynthesis of the corresponding encoded proteins,polypeptides, fragments and variants as recombinant or heterologous geneproducts, (b) as probes for detection and quantification of the nucleicacids disclosed herein, (c) as sequence templates for preparingantisense molecules, and the like. Such uses are described more fully inthe following disclosure.

Furthermore, the proteins and polypeptides described herein, andfragments and variants thereof, may be used, in ways that include (a)serving as an immunogen to stimulate the production of an anti-MCP-1antibody, (b) a capture antigen in an immunogenic assay for such anantibody, (c) as a target for screening for substances that bind to aMCP-1 polypeptide described herein, and (d) a target for a MCP-1specific antibody such that treatment with the antibody affects themolecular and/or cellular function mediated by the target.

In view of its strong effects in modulating cell growth, an increase ofMCP-1 polypeptide expression or activity can be used to promote cellsurvival. Conversely, a decrease in MCP-1 polypeptide expression can beused to induce cell death.

Further embodiments, features, and the like regarding the antibodies ofthe invention are provided in additional detail below.

Sequence Listing

The heavy chain and light chain variable region nucleotide and aminoacid sequences of representative human anti-MCP-1 antibodies areprovided in the sequence listing, the contents of which are summarizedin Table 1 below.

TABLE 1 mAb SEQ ID ID No.: Sequence NO: 1.1.1 Nucleotide sequenceencoding the variable region of the heavy chain 1 Amino acid sequenceencoding the variable region of the heavy chain 2 Nucleotide sequenceencoding the variable region of the light chain 3 Amino acid sequenceencoding the variable region of the light chain 4 1.10.1 Nucleotidesequence encoding the variable region of the heavy chain 5 Amino acidsequence encoding the variable region of the heavy chain 6 Nucleotidesequence encoding the variable region of the light chain 7 Amino acidsequence encoding the variable region of the light chain 8 1.12.1Nucleotide sequence encoding the variable region of the heavy chain 9Amino acid sequence encoding the variable region of the heavy chain 10Nucleotide sequence encoding the variable region of the light chain 11Amino acid sequence encoding the variable region of the light chain 121.13.1 Nucleotide sequence encoding the variable region of the heavychain 13 Amino acid sequence encoding the variable region of the heavychain 14 Nucleotide sequence encoding the variable region of the lightchain 15 Amino acid sequence encoding the variable region of the lightchain 16 1.18.1 Nucleotide sequence encoding the variable region of theheavy chain 17 Amino acid sequence encoding the variable region of theheavy chain 18 Nucleotide sequence encoding the variable region of thelight chain 19 Amino acid sequence encoding the variable region of thelight chain 20 1.2.1 Nucleotide sequence encoding the variable region ofthe heavy chain 21 Amino acid sequence encoding the variable region ofthe heavy chain 22 Nucleotide sequence encoding the variable region ofthe light chain 23 Amino acid sequence encoding the variable region ofthe light chain 24 1.3.1 Nucleotide sequence encoding the variableregion of the heavy chain 25 Amino acid sequence encoding the variableregion of the heavy chain 26 Nucleotide sequence encoding the variableregion of the light chain 27 Amino acid sequence encoding the variableregion of the light chain 28 1.5.1 Nucleotide sequence encoding thevariable region of the heavy chain 29 Amino acid sequence encoding thevariable region of the heavy chain 30 Nucleotide sequence encoding thevariable region of the light chain 31 Amino acid sequence encoding thevariable region of the light chain 32 1.6.1 Nucleotide sequence encodingthe variable region of the heavy chain 33 Amino acid sequence encodingthe variable region of the heavy chain 34 Nucleotide sequence encodingthe variable region of the light chain 35 Amino acid sequence encodingthe variable region of the light chain 36 1.7.1 Nucleotide sequenceencoding the variable region of the heavy chain 37 Amino acid sequenceencoding the variable region of the heavy chain 38 Nucleotide sequenceencoding the variable region of the light chain 39 Amino acid sequenceencoding the variable region of the light chain 40 1.8.1 Nucleotidesequence encoding the variable region of the heavy chain 41 Amino acidsequence encoding the variable region of the heavy chain 42 Nucleotidesequence encoding the variable region of the light chain 43 Amino acidsequence encoding the variable region of the light chain 44 1.9.1Nucleotide sequence encoding the variable region of the heavy chain 45Amino acid sequence encoding the variable region of the heavy chain 46Nucleotide sequence encoding the variable region of the light chain 47Amino acid sequence encoding the variable region of the light chain 482.3.1 Nucleotide sequence encoding the variable region of the heavychain 49 Amino acid sequence encoding the variable region of the heavychain 50 Nucleotide sequence encoding the variable region of the lightchain 51 Amino acid sequence encoding the variable region of the lightchain 52 2.4.1 Nucleotide sequence encoding the variable region of theheavy chain 53 Amino acid sequence encoding the variable region of theheavy chain 54 Nucleotide sequence encoding the variable region of thelight chain 55 Amino acid sequence encoding the variable region of thelight chain 56 3.10.1 Nucleotide sequence encoding the variable regionof the heavy chain 57 Amino acid sequence encoding the variable regionof the heavy chain 58 Nucleotide sequence encoding the variable regionof the light chain 59 Amino acid sequence encoding the variable regionof the light chain 60 3.11.1 Nucleotide sequence encoding the variableregion of the heavy chain 61 Amino acid sequence encoding the variableregion of the heavy chain 62 Nucleotide sequence encoding the variableregion of the light chain 63 Amino acid sequence encoding the variableregion of the light chain 64 3.15.1 Nucleotide sequence encoding thevariable region of the heavy chain 65 Amino acid sequence encoding thevariable region of the heavy chain 66 Nucleotide sequence encoding thevariable region of the light chain 67 Amino acid sequence encoding thevariable region of the light chain 68 3.16.1 Nucleotide sequenceencoding the variable region of the heavy chain 69 Amino acid sequenceencoding the variable region of the heavy chain 70 Nucleotide sequenceencoding the variable region of the light chain 71 Amino acid sequenceencoding the variable region of the light chain 72 3.2 Nucleotidesequence encoding the variable region of the heavy chain 73 Amino acidsequence encoding the variable region of the heavy chain 74 Nucleotidesequence encoding the variable region of the light chain 75 Amino acidsequence encoding the variable region of the light chain 76 3.4.1Nucleotide sequence encoding the variable region of the heavy chain 77Amino acid sequence encoding the variable region of the heavy chain 78Nucleotide sequence encoding the variable region of the light chain 79Amino acid sequence encoding the variable region of the light chain 803.5.1 Nucleotide sequence encoding the variable region of the heavychain 81 Amino acid sequence encoding the variable region of the heavychain 82 Nucleotide sequence encoding the variable region of the lightchain 83 Amino acid sequence encoding the variable region of the lightchain 84 3.6.1 Nucleotide sequence encoding the variable region of theheavy chain 85 Amino acid sequence encoding the variable region of theheavy chain 86 Nucleotide sequence encoding the variable region of thelight chain 87 Amino acid sequence encoding the variable region of thelight chain 88 3.7.1 Nucleotide sequence encoding the variable region ofthe heavy chain 89 Amino acid sequence encoding the variable region ofthe heavy chain 90 Nucleotide sequence encoding the variable region ofthe light chain 91 Amino acid sequence encoding the variable region ofthe light chain 92 3.9 Nucleotide sequence encoding the variable regionof the heavy chain 93 Amino acid sequence encoding the variable regionof the heavy chain 94 Nucleotide sequence encoding the variable regionof the light chain 95 Amino acid sequence encoding the variable regionof the light chain 96 4.4 Nucleotide sequence encoding the variableregion of the heavy chain 97 Amino acid sequence encoding the variableregion of the heavy chain 98 Nucleotide sequence encoding the variableregion of the light chain 99 Amino acid sequence encoding the variableregion of the light chain 100 4.5.1 Nucleotide sequence encoding thevariable region of the heavy chain 101 Amino acid sequence encoding thevariable region of the heavy chain 102 Nucleotide sequence encoding thevariable region of the light chain 103 Amino acid sequence encoding thevariable region of the light chain 104 4.6.1 Nucleotide sequenceencoding the variable region of the heavy chain 105 Amino acid sequenceencoding the variable region of the heavy chain 106 Nucleotide sequenceencoding the variable region of the light chain 107 Amino acid sequenceencoding the variable region of the light chain 108 4.7.1 Nucleotidesequence encoding the variable region of the heavy chain 109 Amino acidsequence encoding the variable region of the heavy chain 110 Nucleotidesequence encoding the variable region of the light chain 111 Amino acidsequence encoding the variable region of the light chain 112 5.3.1Nucleotide sequence encoding the variable region of the heavy chain 113Amino acid sequence encoding the variable region of the heavy chain 114Nucleotide sequence encoding the variable region of the light chain 115Amino acid sequence encoding the variable region of the light chain 1163.1 Nucleotide sequence encoding the variable region of the heavy chain117 Amino acid sequence encoding the variable region of the heavy chain118 Nucleotide sequence encoding the variable region of the light chain119 Amino acid sequence encoding the variable region of the light chain120 1.11.1 Nucleotide sequence encoding the variable region of the heavychain 121 Amino acid sequence encoding the variable region of the heavychain 122 Nucleotide sequence encoding the variable region of the lightchain 123 Amino acid sequence encoding the variable region of the lightchain 124 1.14.1 Nucleotide sequence encoding the variable region of theheavy chain 125 Amino acid sequence encoding the variable region of theheavy chain 126 Nucleotide sequence encoding the variable region of thelight chain 127 Amino acid sequence encoding the variable region of thelight chain 128 1.4.1 Nucleotide sequence encoding the variable regionof the heavy chain 129 Amino acid sequence encoding the variable regionof the heavy chain 130 Nucleotide sequence encoding the variable regionof the light chain 131 Amino acid sequence encoding the variable regionof the light chain 132 3.14.1 Nucleotide sequence encoding the variableregion of the heavy chain 133 Amino acid sequence encoding the variableregion of the heavy chain 134 Nucleotide sequence encoding the variableregion of the light chain 135 Amino acid sequence encoding the variableregion of the light chain 136 3.8 Nucleotide sequence encoding thevariable region of the heavy chain 137 Amino acid sequence encoding thevariable region of the heavy chain 138 Nucleotide sequence encoding thevariable region of the light chain 139 Amino acid sequence encoding thevariable region of the light chain 140 4.8.1 Nucleotide sequenceencoding the variable region of the heavy chain 141 Amino acid sequenceencoding the variable region of the heavy chain 142 Nucleotide sequenceencoding the variable region of the light chain 143 Amino acid sequenceencoding the variable region of the light chain 144 5.1 Nucleotidesequence encoding the variable region of the heavy chain 145 Amino acidsequence encoding the variable region of the heavy chain 146 Nucleotidesequence encoding the variable region of the light chain 147 Amino acidsequence encoding the variable region of the light chain 148Definitions

Unless otherwise defined, scientific and technical terms used inconnection with the invention described herein shall have the meaningsthat are commonly understood by those of ordinary skill in the art.Further, unless otherwise required by context, singular terms shallinclude pluralities and plural terms shall include the singular.Generally, nomenclatures utilized in connection with, and techniques of,cell and tissue culture, molecular biology, and protein and oligo- orpolynucleotide chemistry and hybridization described herein are thosewell known and commonly used in the art. Standard techniques are usedfor recombinant DNA, oligonucleotide synthesis, and tissue culture andtransformation (e.g., electroporation, lipofection). Enzymatic reactionsand purification techniques are performed according to manufacturer'sspecifications or as commonly accomplished in the art or as describedherein. The foregoing techniques and procedures are generally performedaccording to conventional methods well known in the art and as describedin various general and more specific references that are cited anddiscussed throughout the instant application. See, e.g., Sambrook etal., Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. 1989), which is incorporatedherein by reference. The nomenclatures utilized in connection with, andthe laboratory procedures and techniques of, analytical chemistry,synthetic organic chemistry, and medicinal and pharmaceutical chemistrydescribed herein are those well known and commonly used in the art.Standard techniques are used for chemical syntheses, chemical analyses,pharmaceutical preparation, formulation, and delivery, and treatment ofpatients.

As utilized in accordance with the embodiments provided herein, thefollowing terms, unless otherwise indicated, shall be understood to havethe following meanings:

The term “isolated polynucleotide” as used herein shall mean apolynucleotide of genomic, cDNA, or synthetic origin or some combinationthereof, which by virtue of its origin the “isolated polynucleotide” (1)is not associated with all or a portion of a polynucleotide in which the“isolated polynucleotide” is found in nature, (2) is operably linked toa polynucleotide which it is not linked to in nature, or (3) does notoccur in nature as part of a larger sequence.

The term “isolated protein” referred to herein means a protein of cDNA,recombinant RNA, or synthetic origin or some combination thereof, whichby virtue of its origin, or source of derivation, the “isolated protein”(1) is not associated with proteins found in nature, (2) is free ofother proteins from the same source, e.g. free of murine proteins, (3)is expressed by a cell from a different species, or (4) does not occurin nature.

The term “polypeptide” is used herein as a generic term to refer tonative protein, fragments, or analogs of a polypeptide sequence. Hence,native protein, fragments, and analogs are species of the polypeptidegenus. Preferred polypeptides in accordance with the invention comprisethe human heavy chain immunoglobulin molecules and the human kappa lightchain immunoglobulin molecules, as well as antibody molecules formed bycombinations comprising the heavy chain immunoglobulin molecules withlight chain immunoglobulin molecules, such as the kappa light chainimmunoglobulin molecules, and vice versa, as well as fragments andanalogs thereof.

The term “naturally occurring” as used herein as applied to an objectrefers to the fact that an object can be found in nature. For example, apolypeptide or polynucleotide sequence that is present in an organism(including viruses) that can be isolated from a source in nature andwhich has not been intentionally modified by man in the laboratory orotherwise is naturally occurring.

The term “operably linked” as used herein refers to positions ofcomponents so described are in a relationship permitting them tofunction in their intended manner. A control sequence “operably linked”to a coding sequence is ligated in such a way that expression of thecoding sequence is achieved under conditions compatible with the controlsequences.

The term “control sequence” as used herein refers to polynucleotidesequences which are necessary to effect the expression and processing ofcoding sequences to which they are ligated. The nature of such controlsequences differs depending upon the host organism; in prokaryotes, suchcontrol sequences generally include promoter, ribosomal binding site,and transcription termination sequence; in eukaryotes, generally, suchcontrol sequences include promoters and transcription terminationsequence. The term “control sequences” is intended to include, at aminimum, all components whose presence is essential for expression andprocessing, and can also include additional components whose presence isadvantageous, for example, leader sequences and fusion partnersequences.

The term “polynucleotide” as referred to herein means a polymeric formof nucleotides of at least 10 bases in length, either ribonucleotides ordeoxynucleotides or a modified form of either type of nucleotide. Theterm includes single and double stranded forms of DNA.

The term “oligonucleotide” referred to herein includes naturallyoccurring, and modified nucleotides linked together by naturallyoccurring, and non-naturally occurring oligonucleotide linkages.Oligonucleotides are a polynucleotide subset generally comprising alength of 200 bases or fewer. Preferably oligonucleotides are 10 to 60bases in length and most preferably 12, 13, 14, 15, 16, 17, 18, 19, or20 to 40 bases in length. Oligonucleotides are usually single stranded,e.g. for probes; although oligonucleotides may be double stranded, e.g.for use in the construction of a gene mutant. Oligonucleotides of theinvention can be either sense or antisense oligonucleotides.

The term “naturally occurring nucleotides” referred to herein includesdeoxyribonucleotides and ribonucleotides. The term “modifiednucleotides” referred to herein includes nucleotides with modified orsubstituted sugar groups and the like. The term “oligonucleotidelinkages” referred to herein includes oligonucleotides linkages such asphosphorothioate, phosphorodithioate, phosphoroselenoate,phosphorodiselenoate, phosphoroanilothioate, phoshoraniladate,phosphoroamidate, and the like. See e.g., LaPlanche et al. Nucl. AcidsRes. 14:9081 (1986); Stec et al. J. Am. Chem. Soc. 106:6077 (1984);Stein et al. Nucl. Acids Res. 16:3209 (1988); Zon et al. Anti-CancerDrug Design 6:539(1991); Zon et al. Oligonucleotides and Analogues: APractical Approach, pp. 87–108 (F. Eckstein, Ed., Oxford UniversityPress, Oxford England (1991)); Stec et al. U.S. Pat. No. 5,151,510;Uhlmann and Peyman Chemical Reviews 90:543 (1990), the disclosures ofwhich are hereby incorporated by reference. An oligonucleotide caninclude a label for detection, if desired.

The term “selectively hybridize” referred to herein means to detectablyand specifically bind. Polynucleotides, oligonucleotides and fragmentsthereof in accordance with the invention selectively hybridize tonucleic acid strands under hybridization and wash conditions thatminimize appreciable amounts of detectable binding to nonspecificnucleic acids. High stringency conditions can be used to achieveselective hybridization conditions as known in the art and discussedherein. Generally, the nucleic acid sequence homology between thepolynucleotides, oligonucleotides, and fragments of the invention and anucleic acid sequence of interest will be at least 80%, and moretypically with preferably increasing homologies of at least 85%, 90%,95%, 99%, and 100%. Two amino acid sequences are homologous if there isa partial or complete identity between their sequences. For example, 85%homology means that 85% of the amino acids are identical when the twosequences are aligned for maximum matching. Gaps (in either of the twosequences being matched) are allowed in maximizing matching; gap lengthsof 5 or less are preferred with 2 or less being more preferred.Alternatively and preferably, two protein sequences (or polypeptidesequences derived from them of at least 30 amino acids in length) arehomologous, as this term is used herein, if they have an alignment scoreof at more than 5 (in standard deviation units) using the program ALIGNwith the mutation data matrix and a gap penalty of 6 or greater. See M.O. Dayhoff, in Atlas of Protein Sequence and Structure, Vol. 5, 101–110and Supplement 2 to Vol. 5, 1–10 (National Biomedical ResearchFoundation 1972). The two sequences or parts thereof are more preferablyhomologous if their amino acids are greater than or equal to 50%identical when optimally aligned using the ALIGN program. The term“corresponds to” is used herein to mean that a polynucleotide sequenceis homologous (i.e., is identical, not strictly evolutionarily related)to all or a portion of a reference polynucleotide sequence, or that apolypeptide sequence is identical to a reference polypeptide sequence.In contradistinction, the term “complementary to” is used herein to meanthat the complementary sequence is homologous to all or a portion of areference polynucleotide sequence. For illustration, the nucleotidesequence “TATAC” corresponds to a reference sequence “TATAC” and iscomplementary to a “GTATA”.

The following terms are used to describe the sequence relationshipsbetween two or more polynucleotide or amino acid sequences: “referencesequence,” “comparison window,” “sequence identity,” “percentage ofsequence identity,” and “substantial identity”. A “reference sequence”is a defined sequence used as a basis for a sequence comparison; areference sequence may be a subset of a larger sequence, for example, asa segment of a full-length cDNA or gene sequence given in a sequencelisting or may comprise a complete cDNA or gene sequence. Generally, areference sequence is at least 18 nucleotides or 6 amino acids inlength, frequently at least 24 nucleotides or 8 amino acids in length,and often at least 48 nucleotides or 16 amino acids in length. Since twopolynucleotides or amino acid sequences may each (1) comprise a sequence(i.e., a portion of the complete polynucleotide or amino acid sequence)that is similar between the two molecules, and (2) may further comprisea sequence that is divergent between the two polynucleotides or aminoacid sequences, sequence comparisons between two (or more) molecules aretypically performed by comparing sequences of the two molecules over a“comparison window” to identify and compare local regions of sequencesimilarity. A “comparison window,” as used herein, refers to aconceptual segment of at least 18 contiguous nucleotide positions or 6amino acids wherein a polynucleotide sequence or amino acid sequence maybe compared to a reference sequence of at least 18 contiguousnucleotides or 6 amino acid sequences and wherein the portion of thepolynucleotide sequence in the comparison window may comprise additions,deletions, substitutions, and the like (i.e., gaps) of 20 percent orless as compared to the reference sequence (which does not compriseadditions or deletions) for optimal alignment of the two sequences.Optimal alignment of sequences for aligning a comparison window may beconducted by the local homology algorithm of Smith and Waterman, Adv.Appl. Math. 2:482 (1981), by the homology alignment algorithm ofNeedleman and Wunsch, J. Mol. Biol. 48:443 (1970), by the search forsimilarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. (U.S.A.)85:2444 (1988), by computerized implementations of these algorithms(GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics SoftwarePackage Release 7.0, (Genetics Computer Group, 575 Science Dr., Madison,Wis.), Geneworks, or MacVector software packages), or by inspection, andthe best alignment (i.e., resulting in the highest percentage ofhomology over the comparison window) generated by the various methods isselected.

The term “sequence identity” means that two polynucleotide or amino acidsequences are identical (i.e., on a nucleotide-by-nucleotide orresidue-by-residue basis) over the comparison window. The term“percentage of sequence identity” is calculated by comparing twooptimally aligned sequences over the window of comparison, determiningthe number of positions at which the identical nucleic acid base (e.g.,A, T, C, G, U, or I) or residue occurs in both sequences to yield thenumber of matched positions, dividing the number of matched positions bythe total number of positions in the comparison window (i.e., the windowsize), and multiplying the result by 100 to yield the percentage ofsequence identity. The terms “substantial identity” as used hereindenotes a characteristic of a polynucleotide or amino acid sequence,wherein the polynucleotide or amino acid comprises a sequence that hasat least 85 percent sequence identity, preferably at least 90 to 95percent sequence identity, more usually at least 99 percent sequenceidentity as compared to a reference sequence over a comparison window ofat least 18 nucleotide (6 amino acid) positions, frequently over awindow of at least 24–48 nucleotide (8–16 amino acid) positions, whereinthe percentage of sequence identity is calculated by comparing thereference sequence to the sequence which may include deletions oradditions which total 20 percent or less of the reference sequence overthe comparison window. The reference sequence may be a subset of alarger sequence.

As used herein, the twenty conventional amino acids and theirabbreviations follow conventional usage. See Immunology—A Synthesis (2ded., Golub, E. S. and Gren, D. R. eds., Sinauer Associates, Sunderland,Mass. 1991), which is incorporated herein by reference. Stereoisomers(e.g., D-amino acids) of the twenty conventional amino acids, unnaturalamino acids such as α-, α-disubstituted amino acids, N-alkyl aminoacids, lactic acid, and other unconventional amino acids may also besuitable components for polypeptides of the invention described herein.Examples of unconventional amino acids include: 4-hydroxyproline,γ-carboxyglutamate, ε-N,N,N-trimethyllysine, ε-N-acetyllysine,O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine,5-hydroxylysine, σ-N-methylarginine, and other similar amino acids andimino acids (e.g., 4-hydroxyproline). In the polypeptide notation usedherein, the left-hand direction is the amino terminal direction and theright-hand direction is the carboxy-terminal direction, in accordancewith standard usage and convention.

Similarly, unless specified otherwise, the left-hand end ofsingle-stranded polynucleotide sequences is the 5′ end; the left-handdirection of double-stranded polynucleotide sequences is referred to asthe 5′ direction. The direction of 5′ to 3′ addition of nascent RNAtranscripts is referred to as the transcription direction; sequenceregions on the DNA strand having the same sequence as the RNA and whichare 5′ to the 5′ end of the RNA transcript are referred to as “upstreamsequences”; sequence regions on the DNA strand having the same sequenceas the RNA and which are 3′ to the 3′ end of the RNA transcript arereferred to as “downstream sequences”.

As applied to polypeptides, the term “substantial identity” means thattwo peptide sequences, when optimally aligned, such as by the programsGAP or BESTFIT using default gap weights, share at least 80 percentsequence identity, preferably at least 90 percent sequence identity,more preferably at least 95 percent sequence identity, and mostpreferably at least 99 percent sequence identity. Preferably, residuepositions that are not identical differ by conservative amino acidsubstitutions. Conservative amino acid substitutions refer to theinterchangeability of residues having similar side chains. For example,a group of amino acids having aliphatic side chains is glycine, alanine,valine, leucine, and isoleucine; a group of amino acids havingaliphatic-hydroxyl side chains is serine and threonine; a group of aminoacids having amide-containing side chains is asparagine and glutamine; agroup of amino acids having aromatic side chains is phenylalanine,tyrosine, and tryptophan; a group of amino acids having basic sidechains is lysine, arginine, and histidine; and a group of amino acidshaving sulfur-containing side chains is cysteine and methionine.Preferred conservative amino acids substitution groups are:valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine,alanine-valine, glutamic-aspartic, and asparagine-glutamine.

As discussed herein, minor variations in the amino acid sequences ofantibodies or immunoglobulin molecules are contemplated as beingencompassed by the invention described herein, providing that thevariations in the amino acid sequence maintain at least 75%, morepreferably at least 80%, 90%, 95%, and most preferably 99%. Inparticular, conservative amino acid replacements are contemplated.Conservative replacements are those that take place within a family ofamino acids that are related in their side chains. Genetically encodedamino acids are generally divided into families: (1) acidic=aspartate,glutamate; (2) basic=lysine, arginine, histidine; (3) non-polar=alanine,valine, leucine, isoleucine, proline, phenylalanine, methionine,tryptophan; and (4) uncharged polar=glycine, asparagine, glutamine,cysteine, serine, threonine, tyrosine. More preferred families are:serine and threonine are aliphatic-hydroxy family; asparagine andglutamine are an amide-containing family; alanine, valine, leucine andisoleucine are an aliphatic family; and phenylalanine, tryptophan, andtyrosine are an aromatic family. For example, it is reasonable to expectthat an isolated replacement of a leucine with an isoleucine or valine,an aspartate with a glutamate, a threonine with a serine, or a similarreplacement of an amino acid with a structurally related amino acid willnot have a major effect on the binding or properties of the resultingmolecule, especially if the replacement does not involve an amino acidwithin a framework site. Whether an amino acid change results in afunctional peptide can readily be determined by assaying the specificactivity of the polypeptide derivative. Assays are described in detailherein. Fragments or analogs of antibodies or immunoglobulin moleculescan be readily prepared by those of ordinary skill in the art. Preferredamino- and carboxy-termini of fragments or analogs occur near boundariesof functional domains. Structural and functional domains can beidentified by comparison of the nucleotide and/or amino acid sequencedata to public or proprietary sequence databases. Preferably,computerized comparison methods are used to identify sequence motifs orpredicted protein conformation domains that occur in other proteins ofknown structure and/or function. Methods to identify protein sequencesthat fold into a known three-dimensional structure are known. Bowie etal., Science 253:164 (1991). Thus, the foregoing examples demonstratethat those of skill in the art can recognize sequence motifs andstructural conformations that may be used to define structural andfunctional domains in accordance with the invention.

Preferred amino acid substitutions are those which: (1) reducesusceptibility to proteolysis, (2) reduce susceptibility to oxidation,(3) alter binding affinity for forming protein complexes, (4) alterbinding affinities, and (4) confer or modify other physicochemical orfunctional properties of such analogs. Analogs can include variousmuteins of a sequence other than the naturally occurring peptidesequence. For example, single or multiple amino acid substitutions(preferably conservative amino acid substitutions) may be made in thenaturally occurring sequence (preferably in the portion of thepolypeptide outside the domain(s) forming intermolecular contacts. Aconservative amino acid substitution should not substantially change thestructural characteristics of the parent sequence (e.g., a replacementamino acid should not tend to break a helix that occurs in the parentsequence, or disrupt other types of secondary structure thatcharacterizes the parent sequence). Examples of art-recognizedpolypeptide secondary and tertiary structures are described in Proteins,Structures and Molecular Principles (Creighton, ed., W. H. Freeman andCompany, New York 1984); Introduction to Protein Structure (Branden, C.and Tooze, J. eds., Garland Publishing, New York, N.Y. 1991); andThornton et al., Nature 354:105 (1991), which are each incorporatedherein by reference.

The term “polypeptide fragment” as used herein refers to a polypeptidethat has an amino-terminal and/or carboxy-terminal deletion, but wherethe remaining amino acid sequence is identical to the correspondingpositions in the naturally occurring sequence deduced, for example, froma full-length cDNA sequence. Fragments typically are at least 5, 6, 8 or10 amino acids long, preferably at least 14 amino acids long, morepreferably at least 20 amino acids long, usually at least 50 amino acidslong, and even more preferably at least 70 amino acids long. The term“analog” as used herein refers to polypeptides which are comprised of asegment of at least 25 amino acids that has substantial identity to aportion of a deduced amino acid sequence and which has at least one ofthe following properties: (1) specific binding to a MCP-1, undersuitable binding conditions, (2) ability to block appropriate MCP-1binding, or (3) ability to inhibit MCP-1 expressing cell growth in vitroor in vivo. Typically, polypeptide analogs comprise a conservative aminoacid substitution (or addition or deletion) with respect to thenaturally occurring sequence. Analogs typically are at least 20 aminoacids long, preferably at least 50 amino acids long or longer, and canoften be as long as a full-length naturally occurring polypeptide.

Peptide analogs are commonly used in the pharmaceutical industry asnon-peptide drugs with properties analogous to those of the templatepeptide. These types of non-peptide compound are termed “peptidemimetics” or “peptidomimetics.” Fauchere, J. Adv. Drug Res. 15:29(1986); Veber and Freidinger, TINS p. 392 (1985); and Evans et al., J.Med. Chem. 30:1229 (1987), which are incorporated herein by reference.Such compounds are often developed with the aid of computerizedmolecular modeling. Peptide mimetics that are structurally similar totherapeutically useful peptides may be used to produce an equivalenttherapeutic or prophylactic effect. Generally, peptidomimetics arestructurally similar to a paradigm polypeptide (i.e., a polypeptide thathas a biochemical property or pharmacological activity), such as humanantibody, but have one or more peptide linkages optionally replaced by alinkage selected from the group consisting of: —CH₂NH—, —CH₂S—,—CH₂—CH₂—, —CH═CH— (cis and trans), —COCH₂—, —CH(OH)CH₂—, and —CH₂SO—,by methods well known in the art. Systematic substitution of one or moreamino acids of a consensus sequence with a D-amino acid of the same type(e.g., D-lysine in place of L-lysine) may be used to generate morestable peptides. In addition, constrained peptides comprising aconsensus sequence or a substantially identical consensus sequencevariation may be generated by methods known in the art (Rizo andGierasch Ann. Rev. Biochem. 61:387 (1992), incorporated herein byreference); for example, by adding internal cysteine residues capable offorming intramolecular disulfide bridges which cyclize the peptide.

“Antibody” or “antibody peptide(s)” refer to an intact antibody, or abinding fragment thereof that competes with the intact antibody forspecific binding. Binding fragments are produced by recombinant DNAtechniques, or by enzymatic or chemical cleavage of intact antibodies.Binding fragments include Fab, Fab′, F(ab′)₂, Fv, and single-chainantibodies. An antibody other than a “bispecific” or “bifunctional”antibody is understood to have each of its binding sites identical. Anantibody substantially inhibits adhesion of a receptor to acounterreceptor when an excess of antibody reduces the quantity ofreceptor bound to counterreceptor by at least about 20%, 40%, 60% or80%, and more usually greater than about 85% (as measured in an in vitrocompetitive binding assay).

The term “epitope” includes any protein determinant capable of specificbinding to an immunoglobulin or T-cell receptor. Epitopic determinantsusually consist of chemically active surface groupings of molecules suchas amino acids or sugar side chains and usually have specificthree-dimensional structural characteristics, as well as specific chargecharacteristics. An antibody is said to specifically bind an antigenwhen the dissociation constant is ≦1 μM, preferably ≦100 nM and mostpreferably ≦10 nM.

The term “agent” is used herein to denote a chemical compound, a mixtureof chemical compounds, a biological macromolecule, or an extract madefrom biological materials.

“Active” or “activity” for the purposes herein refers to form(s) ofMCP-1 polypeptide which retain a biological and/or an immunologicalactivity of native or naturally occurring MCP-1 polypeptides, wherein“biological” activity refers to a biological function (either inhibitoryor stimulatory) caused by a native or naturally occurring MCP-1polypeptide other than the ability to induce the production of anantibody against an antigenic epitope possessed by a native or naturallyoccurring MCP-1 polypeptide and an “immunological” activity refers tothe ability to induce the production of an antibody against an antigenicepitope possessed by a native or naturally occurring MCP-1 polypeptide.

“Treatment” refers to both therapeutic treatment and prophylactic orpreventative measures, wherein the object is to prevent or slow down(lessen) the targeted pathologic condition or disorder. Those in need oftreatment include those already with the disorder as well as those proneto have the disorder or those in whom the disorder is to be prevented.

“Mammal” refers to any animal classified as a mammal, including humans,other primates, such as monkeys, chimpanzees and gorillas, domestic andfarm animals, and zoo, sports, laboratory, or pet animals, such as dogs,cats, cattle, horses, sheep, pigs, goats, rabbits, rodents, etc. Forpurposes of treatment, the mammal is preferably human.

“Carriers” as used herein include pharmaceutically acceptable carriers,excipients, or stabilizers which are nontoxic to the cell or mammalbeing exposed thereto at the dosages and concentrations employed. Oftenthe physiologically acceptable carrier is an aqueous pH bufferedsolution. Examples of physiologically acceptable carriers includebuffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid; low molecular weight (less thanabout 10 residues) polypeptide; proteins, such as serum albumin,gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose or dextrins; chelatingagents such as EDTA; sugar alcohols such as mannitol or sorbitol;salt-forming counterions such as sodium; and/or nonionic surfactantssuch as TWEEN™, polyethylene glycol (PEG), and PLURONICS™.

Papain digestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, each with a single antigen-bindingsite, and a residual “Fc” fragment, a designation reflecting the abilityto crystallize readily. Pepsin treatment yields an “F(ab′)₂” fragmentthat has two antigen-combining sites and is still capable ofcross-linking antigen.

“Fv” is the minimum antibody fragment that contains a completeantigen-recognition and binding site of the antibody. This regionconsists of a dimer of one heavy- and one light-chain variable domain intight, non-covalent association. It is in this configuration that thethree CDRs of each variable domain interact to define an antigen-bindingsite on the surface of the VH-VL dimer. Collectively, the six CDRsconfer antigen-binding specificity to the antibody. However, forexample, even a single variable domain (e.g., the VH or VL portion ofthe Fv dimer or half of an Fv comprising only three CDRs specific for anantigen) may have the ability to recognize and bind antigen, although,possibly, at a lower affinity than the entire binding site.

A Fab fragment also contains the constant domain of the light chain andthe first constant domain (CH1) of the heavy chain. Fab fragments differfrom Fab′ fragments by the addition of a few residues at the carboxyterminus of the heavy chain CH1 domain including one or more cysteinesfrom the antibody hinge region. F(ab′)₂ antibody fragments originallywere produced as pairs of Fab′ fragments which have hinge cysteinesbetween them. Other chemical couplings of antibody fragments are alsoknown.

“Solid phase” means a non-aqueous matrix to which the antibodiesdescribed herein can adhere. Examples of solid phases encompassed hereininclude those formed partially or entirely of glass (e.g., controlledpore glass), polysaccharides (e.g., agarose), polyacrylamides,polystyrene, polyvinyl alcohol and silicones. In certain embodiments,depending on the context, the solid phases can comprise the well of anassay plate; in others it is a purification column (e.g., an affinitychromatography column). This term also includes a discontinuous solidphase of discrete particles, such as those described in U.S. Pat. No.4,275,149.

The term “liposome” is used herein to denote a small vesicle composed ofvarious types of lipids, phospholipids and/or surfactant which is usefulfor delivery of a drug (such as a MCP-1 polypeptide or antibody thereto)to a mammal. The components of the liposomes are commonly arranged in abilayer formation, similar to the lipid arrangement of biologicalmembranes.

The term “small molecule” is used herein to describe a molecule with amolecular weight below about 500 Daltons.

As used herein, the terms “label” or “labeled” refers to incorporationof a detectable marker, e.g., by incorporation of a radiolabeled aminoacid or attachment to a polypeptide of biotinyl moieties that can bedetected by marked avidin (e.g., streptavidin containing a fluorescentmarker or enzymatic activity that can be detected by optical orcolorimetric methods). In certain situations, the label or marker canalso be therapeutic. Various methods of labeling polypeptides andglycoproteins are known in the art and may be used. Examples of labelsfor polypeptides include, but are not limited to, the following:radioisotopes or radionuclides (e.g., ³H, ¹⁴C, ¹⁵N, ³⁵S, ⁹⁰Y, ⁹⁹Tc,¹¹¹In, ¹²⁵I, ¹³¹I), fluorescent labels (e.g., FITC, rhodamine,lanthanide phosphors), enzymatic labels (e.g., horseradish peroxidase,β-galactosidase, luciferase, alkaline phosphatase), chemiluminescent,biotinyl groups, predetermined polypeptide epitopes recognized by asecondary reporter (e.g., leucine zipper pair sequences, binding sitesfor secondary antibodies, metal binding domains, epitope tags). In someembodiments, labels are attached by spacer arms of various lengths toreduce potential steric hindrance.

The term “pharmaceutical agent or drug” as used herein refers to achemical compound or composition capable of inducing a desiredtherapeutic effect when properly administered to a patient. Otherchemistry terms herein are used according to conventional usage in theart, as exemplified by The McGraw-Hill Dictionary of Chemical Terms(Parker, S., Ed., McGraw-Hill, San Francisco (1985)), incorporatedherein by reference).

As used herein, “substantially pure” means an object species is thepredominant species present (i.e., on a molar basis it is more abundantthan any other individual species in the composition), and preferably asubstantially purified fraction is a composition wherein the objectspecies comprises at least about 50 percent (on a molar basis) of allmacromolecular species present. Generally, a substantially purecomposition will comprise more than about 80 percent of allmacromolecular species present in the composition, more preferably morethan about 85%, 90%, 95%, and 99%. Most preferably, the object speciesis purified to essential homogeneity (contaminant species cannot bedetected in the composition by conventional detection methods) whereinthe composition consists essentially of a single macromolecular species.

The term “patient” includes human and veterinary subjects.

Antibody Structure

The basic antibody structural unit is known to comprise a tetramer. Eachtetramer is composed of two identical pairs of polypeptide chains, eachpair having one “light” (about 25 kDa) and one “heavy” chain (about 50to 70 kDa). The amino-terminal portion of each chain includes a variableregion of about 100 to 110 or more amino acids primarily responsible forantigen recognition. The carboxy-terminal portion of each chain definesa constant region primarily responsible for effector function. Humanlight chains are classified as kappa and lambda light chains. Heavychains are classified as mu, delta, gamma, alpha, or epsilon, and definethe antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively.Within light and heavy chains, the variable and constant regions arejoined by a “J” region of about 12 or more amino acids, with the heavychain also including a “D” region of about 10 more amino acids. Seegenerally, Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. RavenPress, N.Y. (1989)) (incorporated by reference in its entirety for allpurposes). The variable regions of each light/heavy chain pair form theantibody-binding site. Thus, an intact antibody has two binding sites.Except in bifunctional or bispecific antibodies, the two binding sitesare the same.

The chains all exhibit the same general structure of relativelyconserved framework regions (FR) joined by three hyper variable regions,also called complementarity determining regions or CDRs. The CDRs fromthe two chains of each pair are aligned by the framework regions,enabling binding to a specific epitope. From N-terminal to C-terminal,both light and heavy chains comprise the domains FR1, CDR1, FR2, CDR2,FR3, CDR3 and FR4. The assignment of amino acids to each domain is inaccordance with the definitions of Kabat, Sequences of Proteins ofImmunological Interest (National Institutes of Health, Bethesda, Md.1991) (1987), or Chothia and Lesk, J. Mol. Biol. 196:901–17 (1987);Chothia et al., Nature 342:878–83 (1989).

A bispecific or bifunctional antibody is an artificial hybrid antibodyhaving two different heavy/light chain pairs and two different bindingsites. Bispecific antibodies can be produced by a variety of methodsincluding fusion of hybridomas or linking of Fab′ fragments. See, e.g.,Songsivilai and Lachmann, Clin. Exp. Immunol. 79: 315–21 (1990);Kostelny et al., J. Immunol. 148:1547–53 (1992). Production ofbispecific antibodies can be a relatively labor intensive processcompared with production of conventional antibodies and yields anddegree of purity are generally lower for bispecific antibodies.Bispecific antibodies do not exist in the form of fragments having asingle binding site (e.g., Fab, Fab′, and Fv).

Human Antibodies and Humanization of Antibodies

Human antibodies avoid certain of the problems associated withantibodies that possess murine or rat variable and/or constant regions.The presence of such murine or rat derived proteins can lead to therapid clearance of the antibodies or can lead to the generation of animmune response against the antibody by a patient. In order to avoid theutilization of murine or rat derived antibodies, fully human antibodiescan be generated through the introduction of human antibody functioninto a rodent so that the rodent produces fully human antibodies.

Human Antibodies

One method for generating fully human antibodies is through the use ofXenoMouse® strains of mice that have been engineered to contain humanheavy chain and light chain genes within their genome. For example, aXenoMouse® mouse containing 245 kb and 190 kb-sized germlineconfiguration fragments of the human heavy chain locus and kappa lightchain locus is described in Green et al., Nature Genetics 7:13–21(1994). The work of Green et al. was extended to the introduction ofgreater than approximately 80% of the human antibody repertoire throughutilization of megabase-sized, germline configuration YAC fragments ofthe human heavy chain loci and kappa light chain loci, respectively. SeeMendez et al., Nature Genetics 15:146–56 (1997) and U.S. patentapplication Ser. No. 08/759,620, filed Dec. 3, 1996, the disclosures ofwhich are hereby incorporated by reference. Further, XenoMouse® micehave been generated that contain the entire lambda light chain locus(U.S. Patent Application Ser. No. 60/334,508, filed Nov. 30, 2001). And,XenoMouse® mice have been generated that produce multiple isotypes (see,e.g., WO 00/76310). XenoMouse® strains are available from Abgenix, Inc.(Fremont, Calif.).

The production of XenoMouse(® mice is further discussed and delineatedin U.S. patent application Ser. No. 07/466,008, filed Jan. 12, 1990,Ser. No. 07/610,515, filed Nov. 8, 1990, Ser. No. 07/919,297, filed Jul.24, 1992, Ser. No. 07/922,649, filed Jul. 30, 1992, filed Ser. No08/031,801, filed Mar. 15, 1993, Ser. No. 08/112,848, filed Aug. 27,1993, Ser. No. 08/234,145, filed Apr. 28, 1994, Ser. No. 08/376,279,filed Jan. 20, 1995, Ser. No. 08/430,938, Apr. 27, 1995, Ser. No.08/464,584, filed Jun. 5, 1995, Ser. No. 08/464,582, filed Jun. 5, 1995,Ser. No. 08/463,191, filed Jun. 5, 1995, Ser. No. 08/462,837, filed Jun.5, 1995, Ser. No. 08/486,853, filed Jun. 5, 1995, Ser. No. 08/486,857,filed Jun. 5, 1995, Ser. No. 08/486,859, filed Jun. 5, 1995, Ser. No.08/462,513, filed Jun. 5, 1995, Ser. No. 08/724,752, filed Oct. 2, 1996,and Ser. No. 08/759,620, filed Dec. 3, 1996 and U.S. Pat. Nos.6,162,963, 6,150,584, 6,114,598, 6,075,181, and 5,939,598 and JapanesePatent Nos. 3 068 180 B2, 3 068 506 B2, and 3 068 507 B2. See alsoMendez et al. Nature Genetics 15:146–156 (1997) and Green and JakobovitsJ. Exp. Med., 188:483–495 (1998). See also European Patent No., EP463,151 B1, grant published Jun. 12, 1996, International PatentApplication No., WO 94/02602, published Feb. 3, 1994, InternationalPatent Application No., WO 96/34096, published Oct. 31, 1996, WO98/24893, published Jun. 11, 1998, WO 00/76310, published Dec. 21, 2000.The disclosures of each of the above-cited patents, applications, andreferences are hereby incorporated by reference in their entirety.

In an alternative approach, others, including GenPharm International,Inc., have utilized a “minilocus” approach. In the minilocus approach,an exogenous Ig locus is mimicked through the inclusion of pieces(individual genes) from the Ig locus. Thus, one or more V_(H) genes, oneor more D_(H) genes, one or more J_(H) genes, a mu constant region, anda second constant region (preferably a gamma constant region) are formedinto a construct for insertion into an animal. This approach isdescribed in U.S. Pat. No. 5,545,807 to Surani et al. and U.S. Pat. Nos.5,545,806, 5,625,825, 5,625,126, 5,633,425, 5,661,016, 5,770,429,5,789,650, 5,814,318, 5,877,397, 5,874,299, and 6,255,458 each toLonberg and Kay, U.S. Pat. Nos. 5,591,669 and 6,023,010 to Krimpenfortand Berns, U.S. Pat. Nos. 5,612,205, 5,721,367, and 5,789,215 to Bernset al., and U.S. Pat. No. 5,643,763 to Choi and Dunn, and GenPharmInternational U.S. patent application Ser. No. 07/574,748, filed Aug.29, 1990, Ser. No. 07/575,962, filed Aug. 31, 1990, Ser. No. 07/810,279,filed Dec. 17, 1991, Ser. No. 07/853,408, filed Mar. 18, 1992, Ser. No.07/904,068, filed Jun. 23, 1992, Ser. No. 07/990,860, filed Dec. 16,1992, Ser. No. 08/053,131, filed Apr. 26, 1993, Ser. No. 08/096,762,filed Jul. 22, 1993, Ser. No. 08/155,301, filed Nov. 18, 1993, Ser. No.08/161,739, filed Dec. 3, 1993, Ser. No. 08/165,699, filed Dec. 10,1993, Ser. No. 08/209,741, filed Mar. 9, 1994, the disclosures of whichare hereby incorporated by reference. See also European Patent No.546,073 B1, International Patent Application Nos. WO 92/03918, WO92/22645, WO 92/22647, WO 92/22670, WO 93/12227, WO 94/00569, WO94/25585, WO 96/14436, WO 97/13852, and WO 98/24884 and U.S. Pat. No.5,981,175, the disclosures of which are hereby incorporated by referencein their entirety. See further Taylor et al., (1992), Chen et al.,(1993), Tuaillon et al., (1993), Choi et al., (1993), Lonberg et al.,(1994), Taylor et al., (1994), and Tuaillon et al., (1995), Fishwild etal., (1996), the disclosures of which are hereby incorporated byreference in their entirety.

Kirin has demonstrated the generation of human antibodies from mice inwhich, through microcell fusion, large pieces of chromosomes, or entirechromosomes, have been introduced. See European Patent Application Nos.773,288 and 843,961, the disclosures of which are hereby incorporated byreference.

Lidak Pharmaceuticals (now Xenorex) has also demonstrated the generationof human antibodies in SCID mice modified by injection of non-malignantmature peripheral leukocytes from a human donor. The modified miceexhibit an immune response characteristic of the human donor uponstimulation with an immunogen, which consists of the production of humanantibodies. See U.S. Pat. Nos. 5,476,996 and 5,698,767, the disclosuresof which are herein incorporated by reference.

Human anti-mouse antibody (HAMA) responses have led the industry toprepare chimeric or otherwise humanized antibodies. While chimericantibodies have a human constant region and a murine variable region, itis expected that certain human anti-chimeric antibody (HACA) responseswill be observed, particularly in chronic or multi-dose utilizations ofthe antibody. Thus, it would be desirable to provide fully humanantibodies against MCP-1 in order to vitiate concerns and/or effects ofHAMA or HACA response.

Humanization and Display Technologies

As discussed above in connection with human antibody generation, thereare advantages to producing antibodies with reduced immunogenicity. To adegree, this can be accomplished in connection with techniques ofhumanization and display techniques using appropriate libraries. It willbe appreciated that murine antibodies or antibodies from other speciescan be humanized or primatized using techniques well known in the art.See e.g., Winter and Harris, Immunol Today 14:43–46 (1993) and Wright etal., Crit, Reviews in Immunol. 12:125–168 (1992). The antibody ofinterest may be engineered by recombinant DNA techniques to substitutethe CH1, CH2, CH3, hinge domains, and/or the framework domain with thecorresponding human sequence (see WO 92/02190 and U.S. Pat. Nos.5,530,101, 5,585,089, 5,693,761, 5,693,792, 5,714,350, and 5,777,085).Also, the use of Ig cDNA for construction of chimeric immunoglobulingenes is known in the art (Liu et al., P.N.A.S. 84:3439 (1987) and J.Immunol. 139:3521 (1987)). mRNA is isolated from a hybridoma or othercell producing the antibody and used to produce cDNA. The cDNA ofinterest may be amplified by the polymerase chain reaction usingspecific primers (U.S. Pat. Nos. 4,683,195 and 4,683,202).Alternatively, a library is made and screened to isolate the sequence ofinterest. The DNA sequence encoding the variable region of the antibodyis then fused to human constant region sequences. The sequences of humanconstant regions genes may be found in Kabat et al., “Sequences ofProteins of Immunological Interest,” N.I.H. publication no. 91-3242(1991). Human C region genes are readily available from known clones.The choice of isotype will be guided by the desired effector functions,such as complement fixation, or activity in antibody-dependent cellularcytotoxicity. Preferred isotypes are IgG1, IgG3 and IgG4. Either of thehuman light chain constant regions, kappa or lambda, may be used. Thechimeric, humanized antibody is then expressed by conventional methods.

Antibody fragments, such as Fv, F(ab′).sub.2 and Fab may be prepared bycleavage of the intact protein, e.g., by protease or chemical cleavage.Alternatively, a truncated gene is designed. For example, a chimericgene encoding a portion of the F(ab′)₂ fragment would include DNAsequences encoding the CH1 domain and hinge region of the H chain,followed by a translational stop codon to yield the truncated molecule.

Consensus sequences of H and L J regions may be used to designoligonucleotides for use as primers to introduce useful restrictionsites into the J region for subsequent linkage of V region segments tohuman C region segments. C region cDNA can be modified by site directedmutagenesis to place a restriction site at the analogous position in thehuman sequence.

Expression vectors include plasmids, retroviruses, YACs, EBV derivedepisomes, and the like. A convenient vector is one that encodes afunctionally complete human CH or CL immunoglobulin sequence, withappropriate restriction sites engineered so that any VH or VL sequencecan be easily inserted and expressed. In such vectors, splicing usuallyoccurs between the splice donor site in the inserted J region and thesplice acceptor site preceding the human C region, and also at thesplice regions that occur within the human CH exons. Polyadenylation andtranscription termination occur at native chromosomal sites downstreamof the coding regions. The resulting chimeric antibody may be joined toany strong promoter, including retroviral LTRs, e.g., SV-40 earlypromoter, (Okayama et al., Mol. Cell. Bio. 3:280 (1983)), Rous sarcomavirus LTR (Gorman et al., P.N.A.S. 79:6777 (1982)), and moloney murineleukemia virus LTR (Grosschedl et al., Cell 41:885 (1985)). Also, aswill be appreciated, native Ig promoters and the like may be used.

Further, human antibodies or antibodies from other species can begenerated through display-type technologies, including, withoutlimitation, phage display, retroviral display, ribosomal display, andother techniques, using techniques well known in the art and theresulting molecules can be subjected to additional maturation, such asaffinity maturation, as such techniques are well known in the art.Wright and Harris, supra., Hanes and Plucthau, PNAS USA 94:4937–4942(1997) (ribosomal display), Parmley and Smith, Gene 73:305–318 (1988)(phage display), Scott, TIBS 17:241–245 (1992), Cwirla et al., PNAS USA87:6378–6382 (1990), Russel et al., Nucl. Acids Res. 21:1081–1085(1993), Hoganboom et al., Immunol. Reviews 130:43–68 (1992), Chiswelland McCafferty, TIBTECH 10:80–84 (1992), and U.S. Pat. No. 5,733,743. Ifdisplay technologies are utilized to produce antibodies that are nothuman, such antibodies can be humanized as described above.

Using these techniques, antibodies can be generated against MCP-1expressing cells, MCP-1 itself, forms of MCP-1, epitopes or peptidesthereof, and expression libraries thereto (see, e.g., U.S. Pat. No.5,703,057) which can thereafter be screened as described above for theactivities described above.

Preparation of Antibodies

Antibodies in accordance with the invention were prepared through theutilization of the XenoMouse® technology, as described below. Such mice,then, are capable of producing human immunoglobulin molecules andantibodies and are deficient in the production of murine immunoglobulinmolecules and antibodies. Technologies utilized for achieving the sameare disclosed in the patents, applications, and references disclosed inthe Background, herein. In particular, however, a preferred embodimentof transgenic production of mice and antibodies therefrom is disclosedin U.S. patent application Ser. No. 08/759,620, filed Dec. 3, 1996 andInternational Patent Application Nos. WO 98/24893, published Jun. 11,1998 and WO 00/76310, published Dec. 21, 2000, the disclosures of whichare hereby incorporated by reference. See also Mendez et al., NatureGenetics 15:146–156 (1997), the disclosure of which is herebyincorporated by reference.

Antibodies, as described herein, are neutralizing high affinityantibodies to human MCP-1. Further, in some embodiments, the antibodiescross react with rat MCP-1. Several different methods have been usedhistorically to generate monoclonal antibodies or polyclonal antibodiesagainst the N-terminus of human MCP-1. These approaches have includedimmunizing with full length human MCP-1 (hMCP-1) or bovine MCP-1(bMCP-1) (Vieira et al., Braz. J. Med. Biol. Res. 21:1005–1011 (1988)),synthetic peptides of human MCP-1 (1–34 or 1–37) (Visser et al., ActaEndocrinol. 90:90–102 (1979)); Logue et al., J. Immunol. Methods137:159–66 (1991)), and multiple antigenic peptides (MAP) of hMCP-1(1–10), hMCP-1 (9–18) and hMCP-1 (24–37) (Magerlein et al., Drug Res.48:783–87 (1998)). These approaches did not produce antibodies suitablefor human therapeutics. (See section entitled “TherapeuticAdministration and Formulation” herein for therapeutic criteria.) Highaffinity antibodies to hMCP-1 are difficult to make because of B celltolerance to the peptide. However, Bradwell et al., (1999) havedemonstrated that immunization with a mixture of human MCP-1 (1–34) andbovine MCP-1 (1–34) MAPs followed by a mixture of human and bovine MAPstargeting the hMCP-1(51–84) and bMCP-1(51–86) was effective in breakingB-cell tolerance to MCP-1 in a human patient with an inoperableparathyroid tumor.

The approach described herein was designed to overcome B-cell toleranceto hMCP-1 as well as to produce a fully human monoclonal antibodysuitable for therapeutic and diagnostic use. XenoMouse® animals wereimmunized with synthetic peptides of MCP-1 (hMCP-1(1–34) andrMCP-1(1–34)), because synthetic peptides have been successfully used togenerate antibodies specific to endogenous human MCP-1 (Visser et al.,(1979)). Furthermore, because the N-terminus of murine MCP-1 is highlyconserved with human MCP-1 (85% identity) and rat MCP-1 (91%), thecombination of peptides was used as an immunogen to break B-celltolerance to murine MCP-1 through molecular mimicry, thereby allowingthe generation of high affinity human anti-human MCP-1 antibodies. Thesepeptides were both coupled to keyhole limpet hemocyanin and emulsifiedin complete Freund's adjuvant or incomplete Freund's adjuvant to enhancethe immunogenicity of these proteins.

After immunization, lymphatic cells (such as B cells) were recoveredfrom the mice that expressed antibodies, and such recovered cell linesfused with a myeloid-type cell line to prepare immortal hybridoma celllines. Such hybridoma cell lines were screened and selected to identifyhybridoma cell lines that produced antibodies specific to the antigen ofinterest. Herein, the production of multiple hybridoma cell lines thatproduce antibodies specific to MCP-1 is described. Further, acharacterization of the antibodies produced by such cell lines isprovided, including nucleotide and amino acid sequence analyses of theheavy and light chains of such antibodies.

Embodiments of the invention provide for the production of multiplehybridoma cell lines that produce antibodies specific to MCP-1. Furtherembodiments relate to antibodies that bind to and neutralize theactivitiy of othe MCP-1 family members including MCP-2, MCP-3, andMCP-4. The supernatants are also screened for immunoreactivity againstfragments of MCP-1 to further epitope map the different antibodiesagainst related humun chemokines and against rat MCP-1 and the mouseortholog of MCP-1, JE, to determine species cross-reactivity. Furtherembodiments provide a characterization of the antibodies produced bysuch cell lines, including nucleotide and amino acid sequence analysesof the heavy and light chains of such antibodies.

Alternatively, instead of being fused to myeloma cells to generatehybridomas, B cells may be directly assayed. For example, CD19+ B cellsmay be isolated from hyperimmune XenoMouse® mice and allowed toproliferate and differentiate into antibody-secreting plasma cells.Antibodies from the cell supernatants are then screened by ELISA forreactivity against the MCP-1 immunogen. The supernatants are alsoscreened for immunoreactivity against fragments of MCP-1 to furtherepitope map the different antibodies against related human chemokinesand against rat MCP-1 and the mouse ortholog of MCP-1, JE, to determinespecies cross-reactivity. Single plasma cells secreting antibodies withthe desired specificities are then isolated using a MCP-1-specifichemolytic plaque assay (Babcook et al., Proc. Natl. Acad. Sci. USA,93:7843–7848 (1996)). Cells targeted for lysis are preferably sheep redblood cells (SRBCs) coated with the MCP-1 antigen. In the presence of aB cell culture containing plasma cells secreting the immunoglobulin ofinterest and complement, the formation of a plaque indicates specificMCP-1-mediated lysis of the sheep red blood cells surrounding the plasmacell of interest. The single antigen-specific plasma cell in the centerof the plaque can be isolated and the genetic information that encodesthe specificity of the antibody is isolated from the single plasma cell.Using reverse-transcriptase PCR, the DNA encoding the heavy and lightchain variable regions of the antibody can be cloned. Such cloned DNAcan then be further inserted into a suitable expression vector,preferably a vector cassette such as a pcDNA, more preferably such apcDNA vector containing the constant domains of immunglobulin heavy andlight chain. The generated vector can then be transfected into hostcells, preferably CHO cells, and cultured in conventional nutrient mediamodified as appropriate for inducing promoters, selecting transformants,or amplifying the genes encoding the desired sequences. The isolation ofmultiple single plasma cells that produce antibodies specific to MCP-1is described below. Further, the genetic material that encodes thespecificity of the anti-MCP-1 antibody can be isolated, introduced intoa suitable expression vector that can then be transfected into hostcells.

In general, antibodies produced by the fused hybridomas were human IgG2heavy chains with fully human kappa or lambda light chains. In someembodiments, antibodies possess human IgG4 heavy chains as well as IgG2heavy chains. Antibodies may also be of other human isotypes, includingIgG1. The antibodies possessed high affinities, typically possessing aK_(D) of from about 10⁻⁶ through about 10⁻¹² M or below, when measuredby either solid phase and solution phase. Antibodies possessing a K_(D)of at least 10¹¹ M are preferred to inhibit the activity of MCP-1.

Regarding the importance of affinity to therapeutic utility ofanti-MCP-1 antibodies, it will be understood that one can generateanti-MCP-1 antibodies, for example, combinatorially, and assess suchantibodies for binding affinity. One approach that can be utilized is totake the heavy chain cDNA from an antibody, prepared as described aboveand found to have good affinity to MCP-1, and combine it with the lightchain cDNA from a second antibody, prepared as described above and alsofound to have good affinity to MCP-1, to produce a third antibody. Theaffinities of the resulting third antibodies can be measured asdescribed herein and those with desirable dissociation constantsisolated and characterized. Alternatively, the light chain of any of theantibodies described above can be used as a tool to aid in thegeneration of a heavy chain that when paired with the light chain willexhibit a high affinity for MCP-1, or vice versa. These heavy chainvariable regions in this library could be isolated from naive animals,isolated from hyperimmune animals, generated artificially from librariescontaining variable heavy chain sequences that differ in the CDRregions, or generated by any other methods that produce diversity withinthe CDR regions of any heavy chain variable region gene (such as randomor directed mutagenesis). These CDR regions, and in particular CDR3, maybe a significantly different length or sequence identity from the heavychain initially paired with the original antibody. The resulting librarycould then be screened for high affinity binding to MCP-1 to generate atherapeutically relevant antibody molecule with similar properties asthe original antibody (high affinity and neutralization). A similarprocess using the heavy chain or the heavy chain variable region can beused to generate a therapeutically relevant antibody molecule with aunique light chain variable region. Furthermore, the novel heavy chainvariable region, or light chain variable region, can then be used in asimilar fashion as described above to identify a novel light chainvariable region, or heavy chain variable region, that allows thegeneration of a novel antibody molecule.

Another combinatorial approach that can be utilized is to performmutagenesis on germ line heavy and/or light chains that are demonstratedto be utilized in the antibodies in accordance with the inventiondescribed herein, particularly in the complementarity determiningregions (CDRs). The affinities of the resulting antibodies can bemeasured as described herein and those with desirable dissociationconstants isolated and characterized. Upon selection of a preferredbinder, the sequence or sequences encoding the same may be used togenerate recombinant antibodies as described above. Appropriate methodsof performing mutagenesis on an oligonucleotide are known to thoseskilled in the art and include chemical mutagenesis, for example, withsodium bisulfite, enzymatic misincorporation, and exposure to radiation.It is understood that the invention described herein encompassesantibodies with substantial identity, as defined herein, to theantibodies explicitly set forth herein, whether produced by mutagenesisor by any other means. Further, antibodies with conservative ornon-conservative amino acid substitutions, as defined herein, made inthe antibodies explicitly set forth herein, are included in embodimentsof the invention described herein.

Another combinatorial approach that can be used is to express the CDRregions, and in particular CDR3, of the antibodies described above inthe context of framework regions derived from other variable regiongenes. For example, CDR1, CDR2, and CDR3 of the heavy chain of oneanti-MCP-1 antibody could be expressed in the context of the frameworkregions of other heavy chain variable genes. Similarly, CDR1, CDR2, andCDR3 of the light chain of an anti-MCP-1 antibody could be expressed inthe context of the framework regions of other light chain variablegenes. In addition, the germline sequences of these CDR regions could beexpressed in the context of other heavy or light chain variable regiongenes. The resulting antibodies can be assayed for specificity andaffinity and may allow the generation of a novel antibody molecule.

As will be appreciated, antibodies prepared in accordance with theinvention described herein can be expressed in various cell lines.Sequences encoding particular antibodies can be used for transformationof a suitable mammalian host cell. Transformation can be by any knownmethod for introducing polynucleotides into a host cell, including, forexample packaging the polynucleotide in a virus (or into a viral vector)and transducing a host cell with the virus (or vector) or bytransfection procedures known in the art, as exemplified by U.S. Pat.Nos. 4,399,216, 4,912,040, 4,740,461, and 4,959,455 (which patents arehereby incorporated herein by reference). The transformation procedureused depends upon the host to be transformed. Methods for introductionof heterologous polynucleotides into mammalian cells are well known inthe art and include dextran-mediated transfection, calcium phosphateprecipitation, polybrene mediated transfection, protoplast fusion,electroporation, encapsulation of the polynucleotide(s) in liposomes,and direct microinjection of the DNA into nuclei.

Mammalian cell lines available as hosts for expression are well known inthe art and include many immortalized cell lines available from theAmerican Type Culture Collection (ATCC), including but not limited toChinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK)cells, monkey kidney cells (COS), human hepatocellular carcinoma cells(e.g., Hep G2), and a number of other cell lines. Cell lines ofparticular preference are selected through determining which cell lineshave high expression levels and produce antibodies with constitutiveMCP-1 binding properties.

Additional Criteria for Antibody Therapeutics

As discussed herein, the function of the MCP-1 antibody appearsimportant to at least a portion of its mode of operation. The anti-MCP-1antibodies of the instant invention may be made capable of effectorfunction, including complement-dependent cytotoxicity (CDC) andantibody-dependent cellular cytotoxicity (ADCC). There are a number ofisotypes of antibodies that are capable of the same, including, withoutlimitation, the following: murine IgM, murine IgG2a, murine IgG2b,murine IgG3, human IgM, human IgG1, and human IgG3. It will beappreciated that antibodies that are generated need not initiallypossess such an isotype but, rather, the antibody as generated canpossess any isotype and the antibody can be isotype switched thereafterusing conventional techniques that are well known in the art. Suchtechniques include the use of direct recombinant techniques (see, e.g.,U.S. Pat. Nos. 4,816,397 and 6,331,415), cell-cell fusion techniques(see, e.g., U.S. Pat. Nos. 5,916,771 and 6,207,418), among others.

In the cell-cell fusion technique, a myeloma or other cell line isprepared that possesses a heavy chain with any desired isotype andanother myeloma or other cell line is prepared that possesses the lightchain. Such cells can, thereafter, be fused and a cell line expressingan intact antibody can be isolated.

By way of example, the MCP-1 antibodies discussed herein are humananti-MCP-1 IgG2 and IgG4 antibodies. If such antibody possessed desiredbinding to the MCP-1 molecule, it could be readily isotype switched togenerate a human IgM, human IgG1, or human IgG3, IgA1 or IgGA2 isotypes,while still possessing the same variable region (which defines theantibody's specificity and some of its affinity). Such molecule wouldthen be capable of fixing complement and participating in CDC.

Accordingly, as antibody candidates are generated that meet desired“structural” attributes as discussed above, they can generally beprovided with at least certain of the desired “functional” attributesthrough isotype switching.

Epitope Mapping

Immunoblot Analysis

The binding of the antibodies described herein to MCP-1 can be examinedby a number of methods. For example, MCP-1 may be subjected to SDS-PAGEand analyzed by immunoblotting. The SDS-PAGE may be performed either inthe absence or presence of a reduction agent. Such chemicalmodifications may result in the methylation of cysteine residues.Accordingly, it is possible to determine whether the anti-MCP-1antibodies described herein bind to a linear epitope on MCP-1.

Surface-Enhanced Laser Desorption/Ionization (SELDI)

Epitope mapping of the epitope for the MCP-1 antibodies described hereincan also be performed using SELDI. SELDI ProteinChip® arrays are used todefine sites of protein-protein interaction. Antigens are specificallycaptured on antibodies covalently immobilized onto the Protein Chiparray surface by an initial incubation and wash. The bound antigens canbe detected by a laser-induced desorption process and analyzed directlyto determine their mass. Such fragments of the antigen that bind aredesignated as the “epitope” of a protein.

The SELDI process enables individual components within complex molecularcompositions to be detected directly and mapped quantitatively relativeto other components in a rapid, highly-sensitive and scalable manner.SELDI utilizes a diverse array of surface chemistries to capture andpresent large numbers of individual protein molecules for detection by alaser-induced desorption process. The success of the SELDI process isdefined in part by the miniaturization and integration of multiplefunctions, each dependent on different technologies, on a surface(“chip”). SELDI BioChips and other types of SELDI probes are surfaces“enhanced” such that they become active participants in the capture,purification (separation), presentation, detection, and characterizationof individual target molecules (e.g., proteins) or population ofmolecules to be evaluated.

A single SELDI protein BioChip, loaded with only the original sample,can be read thousands of times. The SELDI protein BioChips from LumiCytehold as many as 10,000 addressable protein docking locations per 1square centimeter. Each location may reveal the presence of dozens ofindividual proteins. When the protein composition information from eachlocation is compared and unique information sets combined, the resultingcomposition map reveals an image with sets of features that are usedcollectively to define specific patterns or molecular “fingerprints.”Different fingerprints may be associated with various stages of health,the onset of disease, or the regression of disease associated with theadministration of appropriate therapeutics.

The SELDI process may be described in further detail in four parts.Initially, one or more proteins of interest are captured or “docked” onthe ProteinChip Array, directly from the original source material,without sample preparation and without sample labeling. In a secondstep, the “signal-to-noise” ratio is enhanced by reducing the chemicaland biomolecular “noise.” Such “noise” is reduced through selectiveretention of target on the chip by washing away undesired materials.Further, one or more of the target protein(s) that are captured are readby a rapid, sensitive, laser-induced process (SELDI) that providesdirect information about the target (molecular weight). Lastly, thetarget protein at any one or more locations within the array may becharacterized in situ by performing one or more on-the-chip binding ormodification reactions to characterize protein structure and function.

Phage Display

The epitope for the anti-MCP-1 antibodies described herein can bedetermined by exposing the ProteinChip Array to a combinatorial libraryof random peptide 12-mer displayed on Filamentous phage (New EnglandBiolabs).

Phage display describes a selection technique in which a peptide isexpressed as a fusion with a coat protein of a bacteriophage, resultingin display of the fused protein on the surface of the virion. Panning iscarried out by incubation of a library of phage displayed peptide with aplate or tube coated with the target, washing away the unbound phage,and eluting the specifically bound phage. The eluted phage is thenamplified and taken through additional binding and amplification cyclesto enrich the pool in favor of binding sequences. After three or fourrounds, individual clones binding are further tested for binding byphage ELISA assays performed on antibody-coated wells and characterizedby specific DNA sequencing of positive clones.

After multiple rounds of such panning against the anti-MCP-1 antibodiesdescribed herein, the bound phage may be eluted and subjected to furtherstudies for the identification and characterization of the boundpeptide.

Monoclonal antibodies of the invention were shown to bind importantresidues in the core domain of MCP-1. The neutralizing monoclonalantibodies studied discriminate two functionally important sites inhuman MCP-1, involved with two residues that were previously shown to berequired for binding to the receptor. One site was recognized by alltested antibodies, which competed with the receptor protein for MCP-1binding and involved Arg 24. The second site was detected by the groupof six antibodies that bound the conformational epitope, and theirbinding site appeared to involve Arg24 and Lys35, which are held inclose proximity to the N-terminus by virtue of a disulfide bond betweenC11 and C36.

The MCP-1 variants described herein have been analyzed before withrespect to biological activity, physical receptor binding and structuralintegrity (Jarnagin et al., (1999) Biochemistry 38: 16167–16177;Hemmerich et al, (1999) Biochemistry 38: 13013–13025) and providedvaluable tools in determining the binding epitopes of the antibodies asdescribed below.

Anti MCP-1 antibody 3.11.1 recognizes a conformational epitope anddiffers from other antibodies by its unique sequence of heavy and lightchain, and its ability to cross-react with, and to cross-neutralize,other members of the MCP family, such as MCP-2, MCP-3 and MCP-4. Asshown by the mutagenesis experiments, the binding site of mAb 3.11.1 wasaffected by the change R24A but not by K35A. These data are confirmed bythe Lyc-C on chip digest result with SELDI, which delimits the bindingepitope to be between residues 20–35 of MCP-1.

Determination that the epitope for 3.11.1 is between residues 20–35 wasalso supported by sequence alignment showing that R24, but not K35, wasconserved across other members of the MCP family, specifically MCP-2,MCP-3 and MCP-4. Binding analyses by means of SPOTs peptide synthesizedon membrane (Sigma-Genosys, The Woodlands, Tex.) revealed that bindingsite for at least eight mAbs with linear epitopes involved residues20–25, and included R24. Given the similarities in the results in thesebinding studies and the significant homology between the variable genestructures for all the mAbs binding to linear epitopes on MCP-1, itappears that the antibodies all bind to this neutralizing epitope.

The cluster of the epitope around R24 and K35 explains the neutralizingactivity of all 36 antibodies. The recognized epitope on MCP-1 does notappear to extend to the N-terminal residues up to Pro9. This residueappears to affect receptor signaling, but not binding affinity.

Diagnostic Use

Antibodies prepared in accordance with embodiments of the inventiondescribed herein are useful for assays, particularly in vitro diagnosticassays, for example, for use in determining the level of MCP-1 and allMCP-1 family members in patient samples. The patient samples can be, forexample, bodily fluids, preferably blood, more preferably blood serum,synoival fluid, tissue lysates, and extracts prepared from diseasedtissues. Examples of diagnostic assays include measuring the level ofMCP family chemokines in, for example, human serum, synovial fluid andtissue lysates. Monitoring the level of specific MCP family members maybe used as a surrogate measure of patient response to treatment and as amethod of monitoring the severity of the disease in a patient. Elevatedlevels of MCP-1 compared to levels of other soluble markers wouldindicate the presence of inflammation. The concentration of the MCP-1antigen present in patient samples is determined using a method thatspecifically determines the amount of the antigen that is present. Sucha method includes an ELISA method in which, for example, antibodies ofthe invention may be conveniently immobilized on an insoluble matrix,such as a polymer matrix. Using a population of samples that providesstatistically significant results for each stage of progression ortherapy, a range of concentrations of the antigen that may be consideredcharacteristic of each stage of disease can be designated.

In order to determine the degree of inflammation in a subject understudy, or to characterize the response of the subject to a course oftherapy, a sample of blood is taken from the subject and theconcentration of the MCP-1 antigen present in the sample is determined.The concentration so obtained is used to identify in which range ofconcentrations the value falls. The range so identified correlates witha stage of disease progression or a stage of therapy identified in thevarious populations of diagnosed subjects, thereby providing a stage inthe subject under study.

Gene amplification and/or expression may be measured in a sampledirectly, for example, by conventional Southern blotting, Northernblotting to quantitate the transcription of mRNA (Thomas, Proc. Natl.Acad. Sci. USA, 77:5201–5205 (1980)), dot blotting (DNA analysis), or insitu hybridization, using an appropriately labeled probe, based on thesequences provided herein. Alternatively, antibodies may be employedthat can recognize specific duplexes, including DNA duplexes, RNAduplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes. Theantibodies in turn may be labeled and the assay can be carried out wherethe duplex is bound to a surface, so that upon the formation of duplexon the surface, the presence of antibody bound to the duplex can bedetected.

For example, antibodies, including antibody fragments, can be used toqualitatively or quantitatively detect the expression of MCP-1 proteins.As noted above, the antibody preferably is equipped with a detectable,e.g., fluorescent label, and binding can be monitored by lightmicroscopy, flow cytometry, fluorimetry, or other techniques known inthe art. These techniques are particularly suitable if the amplifiedgene encodes a cell surface protein, e.g., a growth factor. Such bindingassays are performed as known in the art.

In situ detection of antibody binding to the MCP-1 protein can beperformed, for example, by immunofluorescence or immunoelectronmicroscopy. For this purpose, a tissue specimen is removed from thepatient, and a labeled antibody is applied to it, preferably byoverlaying the antibody on a biological sample. This procedure alsoallows for determining the distribution of the marker gene product inthe tissue examined. It will be apparent for those skilled in the artthat a wide variety of histological methods are readily available for insitu detection.

One of the most sensitive and most flexible quantitative methods forquantitating differential gene expression is RT-PCR, which can be usedto compare mRNA levels in different sample populations, in normal andtumor tissues, with or without drug treatment, to characterize patternsof gene expression, to discriminate between closely related mRNAs, andto analyze RNA structure.

The first step in this process is the isolation of mRNA from a targetsample. The starting material is typically total RNA isolated from adisease tissue and corresponding normal tissues, respectively. Thus,mRNA can be extracted, for example, from frozen or archivedparaffin-embedded and fixed (e.g. formalin-fixed) samples of diseasedtissue for comparison with normal tissue of the same type. Methods formRNA extraction are well known in the art and are disclosed in standardtextbooks of molecular biology, including Ausubel et al., CurrentProtocols of Molecular Biology, John Wiley and Sons (1997). Methods forRNA extraction from paraffin embedded tissues are disclosed, forexample, in Rupp and Locker, Lab Invest, 56:A67 (1987), and De Andrés etal., BioTechniques, 18:42044 (1995). In particular, RNA isolation can beperformed using purification kit, buffer set and protease fromcommercial manufacturers, such as Qiagen, according to themanufacturer's instructions. For example, total RNA from cells inculture can be isolated using Qiagen RNeasy mini-columns. Total RNA fromtissue samples can be isolated using RNA Stat-60 (Tel-Test).

As RNA cannot serve as a template for PCR, the first step indifferential gene expression analysis by RT-PCR is the reversetranscription of the RNA template into cDNA, followed by its exponentialamplification in a PCR reaction. The two most commonly used reversetranscriptases are avilo myeloblastosis virus reverse transcriptase(AMV-RT) and Moloney murine leukemia virus reverse transcriptase(MMLV-RT). The reverse transcription step is typically primed usingspecific primers, random hexamers, or oligo-dT primers, depending on thecircumstances and the goal of expression profiling. For example,extracted RNA can be reverse-transcribed using a GeneAmp RNA PCR kit(Perkin Elmer, Calif., USA), following the manufacturer's instructions.The derived cDNA can then be used as a template in the subsequent PCRreaction.

Although the PCR step can use a variety of thermostable DNA-dependentDNA polymerases, it typically employs the Taq DNA polymerase, which hasa 5′–3′ nuclease activity but lacks a 3′–5′ endonuclease activity. Thus,TaqMan PCR typically utilizes the 5′-nuclease activity of Taq or Tthpolymerase to hydrolyze a hybridization probe bound to its targetamplicon, but any enzyme with equivalent 5′ nuclease activity can beused. Two oligonucleotide primers are used to generate anamplicontypical of a PCR reaction. A third oligonucleotide, or probe, isdesigned to detect nucleotide sequence located between the two PCRprimers. The probe is non-extendible by Taq DNA polymerase enzyme, andis labeled with a reporter fluorescent dye and a quencher fluorescentdye. Any laser-induced emission from the reporter dye is quenched by thequenching dye when the two dyes are located close together as they areon the probe. During the amplification reaction, the Taq DNA polymeraseenzyme cleaves the probe in a template-dependent manner. The resultantprobe fragments disassociate in solution, and signal from the releasedreporter dye is free from the quenching effect of the secondfluorophore. One molecule of reporter dye is liberated for each newmolecule synthesized, and detection of the unquenched reporter dyeprovides the basis for quantitative interpretation of the data.

TaqMan RT-PCR can be performed using commercially available equipments,such as, for example, ABI PRIZM 7700TM Sequence Detection System™(Perkin-Elmer-Applied Biosystems, Foster City, Calif., USA), orLightcycler (Roche Molecular Biochemicals, Mannheim, Germany). In apreferred embodiment, the 5′ nuclease procedure is run on a real-timequantitative PCR device such as the ABI PRIZM 7700TM Sequence DetectionSystem™. The system consists of a thermocycler, laser, charge-coupleddevice (CCD), camera and computer. The system amplifies samples in a96-well format on a thermocycler. During amplification, laser-inducedfluorescent signal is collected in real-time through fiber optics cablesfor all 96 wells, and detected at the CCD. The system includes softwarefor running the instrument and for analyzing the data.

5′-Nuclease assay data are initially expressed as Ct, or the thresholdcycle. As discussed above, fluorescence values are recorded during everycycle and represent the amount of product amplified to that point in theamplification reaction. The point when the fluorescent signal is firstrecorded as statistically significant is the threshold cycle (Ct). TheΔCt values are used as quantitative measurement of the relative numberof starting copies of a particular target sequence in a nucleic acidsample when comparing the expression of RNA in a cell from a diseasedtissue with that from a normal cell.

To minimize errors and the effect of sample-to-sample variation, RT-PCRis usually performed using an internal standard. The ideal internalstandard is expressed at a constant level among different tissues, andis unaffected by the experimental treatment. RNAs most frequently usedto normalize patterns of gene expression are mRNAs for the housekeepinggenes glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) and β-actin.

Differential gene expression can also be identified, or confirmed usingthe microarray technique. In this method, nucleotide sequences ofinterest are plated, or arrayed, on a microchip substrate. The arrayedsequences are then hybridized with specific DNA probes from cells ortissues of interest.

In a specific embodiment of the microarray technique, PCR amplifiedinserts of cDNA clones are applied to a substrate in a dense array.Preferably at least 10,000 nucleotide sequences are applied to thesubstrate. The microarrayed genes, immobilized on the microchip at10,000 elements each, are suitable for hybridization under stringentconditions. Fluorescently labeled cDNA probes may be generated throughincorporation of fluorescent nucleotides by reverse transcription of RNAextracted from tissues of interest. Labeled cDNA probes applied to thechip selectively hybridize to each spot of DNA on the array. Afterstringent washing to remove non-specifically bound probes, the chip isscanned by confocal laser microscopy. Quantitation of hybridization ofeach arrayed element allows for assessment of corresponding mRNAabundance. With dual color fluorescence, separately labeled cDNA probesgenerated from two sources of RNA are hybridized pairwise to the array.The relative abundance of the transcripts from the two sourcescorresponding to each specified gene is thus determined simultaneously.The miniaturized scale of the hybridization affords a convenient andrapid evaluation of the expression pattern for large numbers of genes.Such methods have been shown to have the sensitivity required to detectrare transcripts, which are expressed at a few copies per cell, and toreproducibly detect at least approximately two-fold differences in theexpression levels (Schena et al., Proc. Natl. Acad. Sci. USA,93(20)L106–49). The methodology of hybridization of nucleic acids andmicroarray technology is well known in the art.

MCP-1 Agonists and Antagonists

Embodiments of the invention described herein also pertain to variantsof a MCP-1 protein that function as either MCP-1 agonists (mimetics) oras MCP-1 antagonists. Variants of a MCP-1 protein can be generated bymutagenesis, e.g., discrete point mutation or truncation of the MCP-1protein. An agonist of the MCP-1 protein can retain substantially thesame, or a subset of, the biological activities of the naturallyoccurring form of the MCP-1 protein. An antagonist of the MCP-1 proteincan inhibit one or more of the activities of the naturally occurringform of the MCP-1 protein by, for example, competitively binding to adownstream or upstream member of a cellular signaling cascade whichincludes the MCP-1 protein. Thus, specific biological effects can beelicited by treatment with a variant of limited function. In oneembodiment, treatment of a subject with a variant having a subset of thebiological activities of the naturally occurring form of the protein hasfewer side effects in a subject relative to treatment with the naturallyoccurring form of the MCP-1 protein.

Variants of the MCP-1 protein that function as either MCP-1 agonists(mimetics) or as MCP-1 antagonists can be identified by screeningcombinatorial libraries of mutants, e.g., truncation mutants, of theMCP-1 protein for protein agonist or antagonist activity. In oneembodiment, a variegated library of MCP-1 variants is generated bycombinatorial mutagenesis at the nucleic acid level and is encoded by avariegated gene library. A variegated library of MCP-1 variants can beproduced by, for example, enzymatically ligating a mixture of syntheticoligonucleotides into gene sequences such that a degenerate set ofpotential MCP-1 sequences is expressible as individual polypeptides, oralternatively, as a set of larger fusion proteins (e.g., for phagedisplay) containing the set of MCP-1 sequences therein. There are avariety of methods which can be used to produce libraries of potentialMCP-1 variants from a degenerate oligonucleotide sequence. Chemicalsynthesis of a degenerate gene sequence can be performed in an automaticDNA synthesizer, and the synthetic gene then ligated into an appropriateexpression vector. Use of a degenerate set of genes allows for theprovision, in one mixture, of all of the sequences encoding the desiredset of potential MCP-1 variant sequences. Methods for synthesizingdegenerate oligonucleotides are known in the art (see, e.g., Narang,Tetrahedron 39:3 (1983); Itakura et al., Annu. Rev. Biochem. 53:323(1984); Itakura et al, Science 198:1056 (1984); Ike et al., Nucl. AcidRes. 11:477 (1983).

Design and Generation of Other Therapeutics

In accordance with embodiments of the invention described herein andbased on the activity of the antibodies that are produced andcharacterized herein with respect to MCP-1, the design of othertherapeutic modalities beyond antibody moieties is facilitated. Suchmodalities include, without limitation, advanced antibody therapeutics,such as bispecific antibodies, immunotoxins, and radiolabeledtherapeutics, generation of peptide therapeutics, gene therapies,particularly intrabodies, antisense therapeutics, and small molecules.

In connection with the generation of advanced antibody therapeutics,where complement fixation is a desirable attribute, it may be possibleto sidestep the dependence on complement for cell killing through theuse of bispecifics, immunotoxins, or radiolabels, for example.

For example, in connection with bispecific antibodies, bispecificantibodies can be generated that comprise (i) two antibodies one with aspecificity to MCP-1 and another to a second molecule that areconjugated together, (ii) a single antibody that has one chain specificto MCP-1 and a second chain specific to a second molecule, or (iii) asingle chain antibody that has specificity to MCP-1 and the othermolecule. Such bispecific antibodies can be generated using techniquesthat are well known for example, in connection with (i) and (ii) seee.g., Fanger et al. Immunol Methods 4:72–81 (1994) and Wright andHarris, supra. and in connection with (iii) see e.g., Traunecker et al.Int. J. Cancer (Suppl.) 7:51–52 (1992). In each case, the secondspecificity can be made to the heavy chain activation receptors,including, without limitation, CD16 or CD64 (see e.g., Deo et al. 18:127(1997)) or CD89 (see e.g., Valerius et al. Blood 90:4485–4492 (1997)).

In connection with immunotoxins, antibodies can be modified to act asimmunotoxins utilizing techniques that are well known in the art. Seee.g, Vitetta Immunol Today 14:252 (1993). See also U.S. Pat. No.5,194,594. In connection with the preparation of radiolabeledantibodies, such modified antibodies can also be readily preparedutilizing techniques that are well known in the art. See e.g., Junghanset al. in Cancer Chemotherapy and Biotherapy 655–686 (2d edition,Chafner and Longo, eds., Lippincott Raven (1996)). See also U.S. Pat.Nos. 4,681,581, 4,735,210, 5,101,827, 5,102,990 (RE 35,500), 5,648,471,and 5,697,902.

Therapeutic Administration and Formulations

Biologically active anti-MCP-1 antibodies prepared in accordance withthe invention described herein may be used in a sterile pharmaceuticalpreparation or formulation to neutralize the activity of MCP-1 producedin diseased and inflamed tissues, thereby preventing the furtherinfiltration of mononuclear cells into tissues. Such diseased andinflamed tissues occur in many types of human cancer, including breast,ovarian and lung cancer, and in conditions such as glomerulonephritis,artheriosclerosis, and multiple sclerosis. The biologically activeanti-MCP-1 antibody of the instant invention may be employed alone or incombination with other therapeutic agents. For cancer, the anti-MCP-1antibodies may be combined with traditional modes of chemotherapy suchas taxol, doxorubicin, cis-platinum, 5-fluorouracil and other novelinhibitors of the angiogenic process. For treating inflammatory disease,the MCP-1 antibodies may be combined with steroids or antibodies toother cytokines and chemokines that contribute to the disease state.

When used for in vivo administration, the antibody formulation may besterile. This can be readily accomplished by filtration through sterilefiltration membranes, prior to or following lyophilization andreconstitution. The antibody ordinarily will be stored in lyophilizedform or in solution. Therapeutic antibody compositions generally areplaced into a container having a sterile access port, for example, anintravenous solution bag or vial having a stopper pierceable by ahypodermic injection needle.

The route of antibody administration can be in accord with knownmethods, e.g., injection or infusion by intravenous, intraperitoneal,intracerebral, intramuscular, intraocular, intraarterial, intrathecal,inhalation or intralesional routes, or by sustained release systems asnoted below. The antibody is preferably administered continuously byinfusion or by bolus injection.

An effective amount of antibody to be employed therapeutically willdepend, for example, upon the therapeutic objectives, the route ofadministration, and the condition of the patient. Accordingly, it willbe necessary for the therapist to titer the dosage and modify the routeof administration as required to obtain the optimal therapeutic effect.Typically, the clinician will administer antibody until a dosage isreached that achieves the desired effect. The progress of this therapyis easily monitored by conventional assays or by the assays describedherein.

The antibodies of the invention may be prepared in a mixture with apharmaceutically acceptable carrier. This therapeutic composition can beadministered intravenously or through the nose or lung, preferably as aliquid or powder aerosol (lyophilized). The composition may also beadministered parenterally or subcutaneously as desired. Whenadministered systematically, the therapeutic composition should besterile, pyrogen-free and in a parenterally acceptable solution havingdue regard for pH, isotonicity, and stability. These conditions areknown to those skilled in the art. Briefly, dosage formulations of thecompounds of embodiments of the invention described herein are preparedfor storage or administration by mixing the compound having the desireddegree of purity with physiologically acceptable carriers, excipients,or stabilizers. Such materials are non-toxic to the recipients at thedosages and concentrations employed, and include buffers such as TRISHCl, phosphate, citrate, acetate and other organic acid salts;antioxidants such as ascorbic acid; low molecular weight (less thanabout ten residues) peptides such as polyarginine, proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidinone; amino acids such as glycine, glutamic acid,aspartic acid, or arginine; monosaccharides, disaccharides, and othercarbohydrates including cellulose or its derivatives, glucose, mannose,or dextrins; chelating agents such as EDTA; sugar alcohols such asmannitol or sorbitol; counterions such as sodium and/or nonionicsurfactants such as TWEEN, PLURONICS or polyethyleneglycol.

Sterile compositions for injection can be formulated according toconventional pharmaceutical practice as described in Remington'sPharmaceutical Sciences (18^(th) ed, Mack Publishing Company, Easton,Pa. (1990)). For example, dissolution or suspension of the activecompound in a vehicle such as water or naturally occurring vegetable oillike sesame, peanut, or cottonseed oil or a synthetic fatty vehicle likeethyl oleate or the like may be desired. Buffers, preservatives,antioxidants and the like can be incorporated according to acceptedpharmaceutical practice.

Suitable examples of sustained-release preparations includesemipermeable matrices of solid hydrophobic polymers containing thepolypeptide, which matrices are in the form of shaped articles, films ormicrocapsules. Examples of sustained-release matrices includepolyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) asdescribed by Langer et al., J. Biomed Mater. Res., 15:167–277 (1981) andLanger, Chem. Tech., 12:98–105 (1982) or poly(vinylalcohol)),polylactides (U.S. Pat. No. 3,773,919, EP 58,481), copolymers ofL-glutamic acid and gamma ethyl-L-glutamate (Sidman et al., Biopolymers,22:547–556 (1983)), non-degradable ethylene-vinyl acetate (Langer etal., supra), degradable lactic acid-glycolic acid copolymers such as theLUPRON Depot™ (injectable microspheres composed of lactic acid-glycolicacid copolymer and leuprolide acetate), and poly-D-(−)-3-hydroxybutyricacid (EP 133,988).

While polymers such as ethylene-vinyl acetate and lactic acid-glycolicacid enable release of molecules for over 100 days, certain hydrogelsrelease proteins for shorter time periods. When encapsulated proteinsremain in the body for a long time, they may denature or aggregate as aresult of exposure to moisture at 37° C., resulting in a loss ofbiological activity and possible changes in immunogenicity. Rationalstrategies can be devised for protein stabilization depending on themechanism involved. For example, if the aggregation mechanism isdiscovered to be intermolecular S—S bond formation through disulfideinterchange, stabilization may be achieved by modifying sulfhydrylresidues, lyophilizing from acidic solutions, controlling moisturecontent, using appropriate additives, and developing specific polymermatrix compositions.

Sustained-release compositions also include liposomally entrappedantibodies of the invention. Liposomes containing such antibodies areprepared by methods known per se: U.S. Pat. No. DE 3,218,121; Epstein etal., Proc. Natl. Acad. Sci. USA, 82:3688–3692 (1985); Hwang et al.,Proc. Natl. Acad Sci. USA, 77:4030–4034 (1980); EP 52,322; EP 36,676; EP88,046; EP 143,949; 142,641; Japanese patent application 83-118008; U.S.Pat. Nos. 4,485,045 and 4,544,545; and EP 102,324. The dosage of theantibody will be determined by the attending physician taking intoconsideration various factors known to modify the action of drugsincluding severity and type of disease, body weight, sex, diet, time androute of administration, other medications and other relevant clinicalfactors. Therapeutically effective dosages may be determined by eitherin vitro or in vivo methods.

The dosage of the antibody formulation for a given patient will bedetermined by the attending physician taking into consideration variousfactors known to modify the action of drugs including severity and typeof disease, body weight, sex, diet, time and route of administration,other medications and other relevant clinical factors. Therapeuticallyeffective dosages may be determined by either in vitro or in vivomethods.

An effective amount of the antibody of the invention to be employedtherapeutically will depend, for example, upon the therapeuticobjectives, the route of administration, and the condition of thepatient. Accordingly, it will be necessary for the therapist to titerthe dosage and modify the route of administration as required to obtainthe optimal therapeutic effect. A typical daily dosage might range fromabout 0.001 mg/kg to up to 100 mg/kg or more, depending on the factorsmentioned above. Desirable dosage concentrations include 0.001 mg/kg,0.005 mg/kg, 0.01 mg/kg, 0.05 mg/kg, 0.1 mg/kg, 0.5 mg/kg, 1 mg/kg, 5mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 35 mg/kg, 40mg/kg, 45 mg/kg, 50 mg/kg, 55 mg/kg, 60 mg/kg, 65 mg/kg, 70 mg/kg, 75mg/kg, 80 mg/kg, 85 mg/kg, 90 mg/kg, 95 mg/kg, and 100 mg/kg or more.Typically, the clinician will administer the therapeutic antibody untila dosage is reached that achieves the desired effect. The progress ofthis therapy is easily monitored by conventional assays or as describedherein.

EXAMPLES

The following examples, including the experiments conducted and resultsachieved are provided for illustrative purposes only and are not to beconstrued as limiting upon the embodiments of the invention describedherein.

Example 1 MCP-1 Antigen Preparation

The human MCP-1 peptide used as the antigen in these studies had thefollowing amino acid sequence:

QPDAINAPVTCCYNFTNRKISVQRLASYRRITSSK (SEQ ID NO: 149)CPKEAVIFKTIVAKEICADPKQKWVQDSMDHLDKQ TQTPKT

This peptide was expressed recombinantly in E. coli and purchased fromPrepro Tech (Rocky Hill, N.J.).

Example 2 Anti-MCP-1 Antibodies

Antibody Generation

Immunization and selection of animals for harvesting by ELISA.Monoclonal antibodies against MCP-1 were developed by sequentiallyimmunizing XenoMouse® mice (XenoMouse® strains XMG2, XMG4 (3C-1 strain),and a hybrid strain produced through the crossing of XMG2 with an XMG4(3C-1 strain) mouse, Abgenix, Inc. Fremont, Calif.) according to theschedule shown in Table 2. For instance, the initial immunization waswith 10 μg antigen admixed 1:1 v/v with TiterMax Gold. Subsequent boostswere made with 5 or 10 μg antigen admixed 1:1 v/v with 100 μg alum gelin pyrogen-free D-PBS. Some boosts were done with 50% TiterMax Gold,followed by three injections with 10 μg antigen admixed 1:1 v/v with 10μg MCP-1 antigen in alum gel, and then a final boost of 10 μg antigen inPBS. In particular, each mouse was immunized in the footpad bysubcutaneous injection. The animals were immunized on days 0, 4, 7, 10,14, 18, 27, 31, 35 and 42. The animals were bled on days 13 and 26 toobtain sera for harvest selection as described below.

TABLE 2 # of 1^(st) 2^(nd) 3^(rd) 4^(th) 5^(th) 6^(th) 7^(th) 8^(th)9^(th) 10^(th) Fu- Group Strain mice injection boost boost boost Bleedboost boost Bleed boost boost boost boost sion 1 xmg2 7 10 μg/ 5 μg// 5μg/ 5 μg/ 5 μg/ 5 μg/ 10 μg/ 10 μg/ 10 μg/ 10 μg/ mouse mouse mousemouse mouse mouse mouse mouse mouse mouse 2 3C-1 7 10 μg// 5 μg/ 5 μg/ 5μg/ 5 μg/ 5 μg/ 10 μg/ 10 μg/ 10 μg/ 10 μg/ mouse mouse mouse mousemouse mouse mouse mouse mouse mouse 3 (3C-1) × 7 10 μg/ 5 μg/ 5 μg/ 5μg/ 5 μg/ 5 μg/ 10 μg/ 10 μg// 10 μg/ 10 μg/ xmg2 mouse mouse mousemouse mouse mouse mouse mouse mouse mouse TiterMax Alum Alum Alum AlumTiterMax Alum Alum Alum D-PBS Gel Gel Gel Gel Gel Gel Gel Day 0 4 7 1013 14 18 26 27 31 35 42 46

Similarly, other XenoMouse® mice (XenoMouse® strains XMG2 and XMG2L3)were sequentially immunized according to the schedule shown in Table 3.

TABLE 3 # of 1^(st) 2^(nd) Group Strain mice injection boost 3^(rd)boost 4^(th) boost Bleed 5^(th) boost 6^(th) boost Fusion 4 xmg2 4 10μg/ 10 μg/ 10 μg/ 10 μg/ 10 μg/ 10 μg/ mouse mouse mouse mouse mousemouse 5 xmg2L3 4 10 μg/ 10 μg/ 10 μg/ 10 μg/ 10 μg/ 10 μg/ mouse mousemouse mouse mouse mouse TiterMax Alum Alum Gel Alum Gel Alum Gel AlumGel Gel Day 0 3 6 10 13 14 17 21

Anti-MCP-1 antibody titers were determined by indirect ELISA. The titervalue is the reciprocal of the greatest dilution of sera with an ODreading two-fold that of background. Briefly, MCP-1 (84 mer; 1 μg/mL)was coated onto Costar Labcoat Universal Binding Polystyrene 96 wellplates overnight at four degrees. The solution containing unbound MCP-1was removed and the plates were treated with UV light (365 nm) for 4minutes (4000 microjoules). The plates were washed five times with dH₂O.XenoMouse® sera from the MCP-1 immunized animals, or naive XenoMouse®animals, were titrated in 2% milk/PBS at 1:2 dilutions in duplicate froma 1:100 initial dilution. The last well was left The plates were washedfive times with dH₂O.

A goat anti-human IgG Fc-specific HRP-conjugated antibody was added atconcentration of 1 μg/mL for 1 hour at room temperature. The plates werewashed five with dH₂O. The plates were developed with the addition ofTMB for 30 minutes and the ELISA was stopped by the addition of 1 Mphosphoric acid. The specific titer of individual XenoMouse® animals wasdetermined from the optical density at 450 nm and is shown in Tables 4,5, 6, 7, and 8. The titer represents the reciprocal dilution of theserum and therefore the higher the number the greater the humoral immuneresponse to MCP-1. Lymph nodes from all immunized XenoMouse® animalswere harvested for fusion.

TABLE 4 Group 1, footpad, xmg2, 7 mice bleed of Day 13 bleed of Day 26fusion of Day 46 After 4 injections After 6 injections After 10injections Reactivity to MCP-1 Mouse ID Titers via hIgG N160-1 1,00073,000 300,000 N160-2 6,500 600,000 600,000 N160-3 2,300 250,000 125,000N160-4 1,400 125,000 75,000 N160-5 4,000 200,000 225,000 N160-6 2502,400 18,000 N160-7 60 1,600 35,000 NC 175 <100 200

TABLE 5 Group 2, footpad, 3c-1, 7 mice bleed of Day 13 fusion of Day 46After 6 injections After 10 injections Reactivity to MCP-1 Mouse IDTiters via hIgG M724-1 35,000 24,000 M724-3 8,000 7,500 M724-5 8,00020,000 N600-4 9,000 7,500 N600-5 1,800 75,000 N600-6 2,200 20,000 N600-7800 25,000 NC <100 <100

TABLE 6 Group 3, footpad, 3c-1/xmg2 (F1), 7 mice bleed of Day 13 bleedof Day 26 fusion of Day 46 After 4 injections After 6 injections After10 injections Reactivity to MCP-1 Mouse ID Titers via hIgG M219-1 502,200 8,000 M219-2 <100 9,000 18,000 M246-3 800 7,000 18,000 M246-5 85018,000 65,000 M246-9 <100 18,000 55,000 M344-6 <100 800 12,000 M344-10<100 6,000 25,000 NC 200 225 175

TABLE 7 Group 4, XMG2, footpad, 4 mice Capture: bleed of Day 13 bleed ofDay 21 after 4 injections after 6 injections Human Human Human HumanMCP-1 MCP-1 MCP-1 MCP-1 Reactivity Reactivity Reactivity Reactivity toMCP-1 to MCP-1 to MCP-1 to MCP-1 Mouse ID Titers via hIgG Titers via hLTiters via hIgG Titers via hL N493-1 <100 <100 2,500 <100 N493-2 <100<100 1,000 <100 N493-3 300 <100 4,500 <100 N493-4 800 <100 10,000 <100NC 900 100 600 <100 *PC 8,000 3,000

TABLE 8 Group 5, XMG2L3, footpad, 4 mice Capture: bleed after 4injections bleed of after 6 injections Human Human Human Human MCP-1MCP-1 MCP-1 MCP-1 Reactivity Reactivity Reactivity Reactivity to MCP-1to MCP-1 to MCP-1 to MCP-1 Mouse ID Titers via hIgG Titers via hL Titersvia hIgG Titers via hL N259-12 300 300 2,000 700 N259-14 100 400 2,500650 N269-2 700 200 2,800 500 N263-3 900 900 24,000 8,000 NC 900 100 600<100 *PC 8,000 3,000 *For Tables 4–8, NC (negative control) = XMG2 KLHgroup 1, footpad L627-6 PC (positive control) = XMG2 MCP-1 group 1,footpad N160-1

Recovery of lymphocytes, B-cell isolations, fusions and generation ofhybridomas. Immunized mice were sacrificed by cervical dislocation, andthe lymph nodes harvested and pooled from each cohort. The lymphoidcells were dissociated by grinding in DMEM to release the cells from thetissues and the cells were suspended in DMEM. The cells were counted,and 0.9 mL DMEM per 100 million lymphocytes added to the cell pellet toresuspend the cells gently but completely. Using 100 μL of CD90⁺magnetic beads per 100 million cells, the cells were labeled byincubating the cells with the magnetic beads at 4° C. for 15 minutes.The magnetically labeled cell suspension containing up to 10⁸ positivecells (or up to 2×10⁹ total cells) was loaded onto a LS⁺ column and thecolumn washed with DMEM. The total effluent was collected as theCD90-negative fraction (most of these cells are B cells).

P3 myeloma cells and B cell-enriched lymph node cells were combined in aratio of 1:1 (myeloma: lymph nodes) into a 50 mL conical tube in DMEM.The combined cells were centrifuged at 800×g (2000 rpm) for 5–7 minutesand the supernatant immediately removed from the resulting pellet. Twoto four mL of Pronase solution (CalBiochem, Cat. #53702; 0.5 mg/mL inPBS) was added to the cells to resuspend the cell pellet gently. Theenzyme treatment was allowed to proceed for no more than two minutes andthe reaction stopped by the addition of 3–5 mL of FBS. Enough ECFsolution was added to bring the total volume to 40 mL and the mixturewas centrifuged at 800×g (2000 rpm) for 5–7 minutes. The supernatant wasremoved and the cell pellet gently resuspended with a small volume ofECF solution, followed by enough ECF solution to make a total volume of40 mL. The cells were mixed well and counted, then centrifuged at 800×g(2000 rpm) for 5–7 minutes. The supernatant was removed and the cellsresuspended in a small volume of ECF solution. Enough additional ECFsolution was added to adjust the concentration to 2×10⁶ cells/mL.

The cells were then placed in an Electro-Cell-Fusion (ECF) generator(Model ECM2001, Genetronic, Inc., San Diego, Calif.) and fused accordingto the manufacturer's instructions. After ECF, the cell suspensions werecarefully removed from the fusion chamber under sterile conditions andtransferred into a sterile tube containing the same volume of HybridomaMedium in DMEM. The cells were incubated for 15–30 minutes at 37° C.,then centrifuged at 400×g (1000 rpm) for five minutes. The cells weregently resuspended in a small volume of ½ HA medium (1 bottle of 50×HAfrom Sigma, Cat. #A9666 and 1 liter of Hybridoma Medium) and the volumeadjusted appropriately with more ½ HA medium (based on 5×10⁶ B cells per96-well plate and 200 μL per well). The cells were mixed well andpipetted into 96-well plates and allowed to grow. On day 7 or 10,one-half the medium was removed, and the cells re-fed with ½ HA medium.

Selection of candidate antibodies for ELISA. After 14 days of culture,hybridoma supernatants were screened for MCP-1-specific monoclonalantibodies. The ELISA plates (Fisher, Cat. No. 12-565-136) were coatedwith 50 μl/well of MCP-1 (2 μg/mL) in Coating Buffer (0.1 M CarbonateBuffer, pH 9.6, NaHCO₃8.4 g/L), then incubated at 4° C. overnight. Afterincubation, the plates were washed with Washing Buffer (0.05% Tween 20in PBS) three times. 200 μl/well Blocking Buffer (0.5% BSA, 0.1% Tween20, 0.01% Thimerosal in 1×PBS) were added and the plates incubated atroom temperature for 1 hour. After incubation, the plates were washedwith Washing Buffer three times. 50 μL/well of hybridoma supernatants,and positive and negative controls were added and the plates incubatedat room temperature for 2 hours.

The positive control used throughout was XMG2 MCP-1 Group 1, footpadN160-7 and the negative control was XMG2 KLH Group 1, footpad L627-6.After incubation, the plates were washed three times with WashingBuffer. 100 μL/well of detection antibody goat anti-huIgGfc-HRP (Caltag,Cat. #H10507), (and goat anti-hlgkappa-HRP (Southern Biotechnology, Cat.# 2060-05) and goat anti-hlglambda (Southern Biotechnology, Cat. #2070-05) in secondary screening) were added and the plates incubated atroom temperature for 1 hour. In the secondary screen, three sets ofsamples (positives in first screening) were screened, one set for hIgGdetection, one set for hKappa detection, and one set for hlambdadetection. After incubation, the plates were washed three times withWashing Buffer. 100 μL/well of TMB (BioFX Lab. Cat. #TMSK-0100-01) wereadded and the plates allowed to develop for about 10 minutes (untilnegative control wells barely started to show color), then 50 μL/wellstop solution (TMB Stop Solution (BioFX Lab. Cat. #STPR-0100-01) wereadded and the plates read on an ELISA plate reader at wavelength 450 nm.The OD readings from the positive wells are presented in Table 9.

TABLE 9 ELISA IC50 mAb OD- IC50 Ca++ Chemotaxis Affinity Cross- CloneMCP-1 Flux (μg/mL) (μg/mL) (pMol) Reactivity 1.1.1 3.638  0.24 + 0.034 0.27 + 0.034 2.7 1.2.1 3.466  0.18 + 0.008  0.24 + 0.034 77 1.3.1 4 0.12 + 0.012  0.24 + 0.059 55 1.4.1 4  0.11 + 0.005  0.51 + 0.035 961.5.1 0.51  0.21 + 0.027  0.34 + 0.054 4.2 1.6.1 3.918   1 + 0.24  12 +5.8 228 1.7.1 3.521  0.11 + 0.013  0.35 + 0.064 4.9 1.8.1 3.472  0.26 +0.076 0.88 + 0.21 4 1.9.1 3.6561  1.2 + 0.38 35 + 54 96 1.10.1 3.8450.18 + 0.11  1.2 + 0.55 9.6 1.11.1 3.905 0.098 + 0.008 0.81 + 0.24 4.21.12.1 4 0.13 + 0.02  0.35 + 0.039 13 1.13.1 4  0.11 + 0.015  0.5 +0.091 71 1.14.1 2.064 0.41 + 0.1  0.58 + 0.18 6 1.18.1 0.9984  0.18 +0.055 0.29 + 0.07 3.8 2.3.1 3.876  0.14 + 0.021  0.58 + 0.085 96 2.4.13.892 0.26 + 0.18 >5 14 mouse JE 3.2 3.96 ND MCP-2, MCP-3, eotaxin 3.4.13.86  0.24 + 0.019 0.51 + 0.1  45 3.5.1 3.765 0.58 + 0.29 3.1 + 1.1 1003.6.1 3.593 0.17 + 0.04 0.52 + 0.18 15 3.7.1 4 0.094 + 0.023  0.98 +0.019 4.8 3.8.1 3.603  0.27 + 0.028  0.7 + 0.19 3.4 3.10.1 3.634 0.3 +0.1 0.25 + 0.1  90 MCP-2, MCP-3, eotaxin 3.11.1 4 0.092 + 0.023 0.33 +0.47 3.3 MCP-2, MCP-3, MCP-4 eotaxin 3.14.1 4 1.3 + 0.3  1.4 + 0.47 ND3.15.1 4  0.12 + 0.034 0.89 + 0.1  3.4 3.16.1 3.921 0.16 + 0.08  0.4 +0.081 25 4.5.1 3.38  0.27 + 0.074 0.75 + 0.18 61 4.6.1 3.51 0.31 + 0.06 0.4 + 0.056 330 4.7.1 3.843  0.39 + 0.063 0.45 + 0.11 280 4.8.1 40.22 + 0.77  0.29 + 0.032 102 4.9.1 3.415 0.083 + .0094  0.21 + 0.035 ND5.1 4 3.5 + 2.1 1.3 + 1.2 1610 5.2.1 3.714  2.5 + 0.66 2.1 + 1.7 319Rantes 5.3.1 4  1.8 + 0.56  2.6 + 0.31 450 ND = not doneCharacterization of Anti-MCP-1 Antibodies for Biologic Activity.

Neutralization of MCP-1 bioactivity with anti-MCP-1 antibodies—FLIPRassay. DMSO and Pluronic Acid (20% DMSO solution) were added to a vialof Fluo-4 (Molecular Probes) to yield a final concentration of 5 mMFluo4. THP-1 cells were resuspended in prewarmed (37° C.) loading bufferat 3×10 e6/mL and 1 μL of Fluo-4 dye per ml of cells was added to give afinal concentration of dye at 5 μM. The cells were incubated in the darkat 37° C. for 45–50 minutes. After incubation, the cells werecentrifuged at 1000 RPM for 5–10 min. The cells were resuspended inloading buffer and the centrifugation was repeated. The cells wereresuspended at 1.667e6/mL. At a concentration of 200,000 cells/well, thecells were added to a 96-well plate and centrifuged gently. After takinga baseline reading, a second reading was taken upon subsequent additionof 3.5 nM MCP-1 in the presence or absence of varying concentrations ofanti-MCP-1 antibodies. Addition of MCP-1 to the THP-1 cells resulted ina rise of intracellular calcium leading to enhancement of fluorescenceintensity of Fluo-4 dye. Upon addition of increasing concentrations ofneutralizing antibody, the fluorescent dye intensity within the cellswas decreased, thus indicating that the antibody tested wasneutralizing. The concentration of antibody that yielded a 50% decreasein MCP-1 induced fluorescence intensity is presented in Table 9.

Neutralization of MCP-1-induced cell migration. An automated 96-wellchemotaxis assay was developed using THP-1 cells and a Beckman BiomekF/X robotic system. Using a specially designed 96-well plate, a framedfilter with the filter membrane bonded to a rigid frame, the chemotaxisassay was run in a NeuroProbe 96-well disposable microplate with a wellvolume of either 30 μl or 300 μl and pore diameter ranging from 2–14 μm.The Neuroprobe 96-well plate provides bottom wells for placing the MCP-1chemoattractant and other reagents such as anti-MCP-1 antibodies incell-migration assays. No top wells were required because the framedfilter was coated with a hydrophobic mask that confines eachcell-suspension sample to its site on top of the filter.

The optimum conditions for this assay were: 100,000 cells/well with 90min incubation at 37° C. Suspensions of THP-1 cells that had beepre-loaded with dye from Molecular Probes were pipetted directly ontothe sites on the upper side of the filter and incubated at 37° C. for1–2 hours. After incubation, the cells that had migrated to the bottomof the filter and into the microplate were counted by placing themicroplate into an FMAT purchased from Applied Biosystems.

MCP-1 induced cell migration for THP-1 cells and the maximal cellmigration was reached at 1 nM with a signal to noise ratio of 10–15fold. Using either hybridoma supernatants or fresh hybridoma media,MCP-1-dependent migration was detected. The variability of the assay wasminimal (C.V˜15). The number of cells migrating to the bottom of thefilters was decreased in a dose dependent manner when antibodies toMCP-1 were included with the chemoattractant.

Determination of anti-MCP-1 antibody affinity using Biacore analysis.The antibody/MCP-1 interaction analysis was performed at 25° C. usingtwo CM5 chips docked in Biacore 3000 optical biosensors. Individual flowcells on each chip were activated with a 7-minute injection of NHS/EDC,carbohydrazide was coupled through the NHS ester using a 7-minuteinjection, and the residual activated groups were blocked with a7-minute injection of ethanolamine. The monosaccharide residues of eachantibody were oxidized using 1 mM sodium metaperiodate in 100 mM sodiumacetate, pH 5.5 at 4° C. for 30 minutes. The oxidized antibody wasdesalted into 100 mM sodium acetate, pH 5.0, to couple the antibody tothe carbohydrazide-modified surface. The mAb surfaces were stabilized byreducing the hydrazone bond with 0.1 M sodium cyanoborohydride. Theantigen/antibody interaction was tested by injecting 0, 0.049, 0.15,0.4, 1.3, 4 and 12 nM of MCP-1 (Peprotech, N.J.) in running buffer (10mM HEPES, 150 mM NaCl, 0.005% surfactant, 200 μg/ml BSA, pH 7.4). Thesurfaces were regenerated with a 12-second pulse of 15 mM H₃PO₄. Theantigen/antibody interaction was tested by injecting duplicate antigensamples diluted in running buffer (10 mM HEPES, 50 mM NaCl, 0.005%surfactant, 200 μg/mL BSA, pH 7.4), in a 300-fold concentration range.The surfaces were regenerated with a 12-second pulse of 15 mM H₃PO₄. Todetermine the kinetics of each interaction, the data sets were fitglobally to a 1:1 interaction model that included a parameter for masstransport. The calculated affinities of interaction are reported inTable 9.

Determining cross-reactivity of anti-MCP-1 antibodies with otherchemokines. ELISA plates (Fisher Cat. No. 12-565-136) were coated with50 μl/well of MCP-1, MCP-2, MCP-3, MCP-4, RANTES, GRO-alpha, MIP-1alpha, eotaxin, rat MCP-1 and mouse JE (2 μg/ml) in coating buffer (0.1M carbonate buffer, pH 9.6, NaHCO₃ 8.4 g/L, then incubated at 4° C.overnight. After incubation, the plates were washed with washing buffer(0.05% Tween 20 in PBS) three times. 200 μL/well blocking buffer (0.5%BSA, 0.1% Tween 20, 0.01% Thimerosal in 1×PBS) were added and the platesincubated at room temperature for 1 hour. After incubation, the plateswere washed with washing buffer three times. 50 μL/well of hybridomasupernatants, and positive and negative controls (positive control wasanti-MCP-1 antibody purchased from R&D Sciences, and negative controlwas an antibody to Keyhole Limpet Hemocyanin produced at Abgenix) wereadded and the plates incubated at room temperature for 2 hours. Afterincubation, the plates were washed three times with washing buffer. 100μL/well of detection antibody goat anti-huIgGfc-HRP (Caltag, Cat.#H10507), (goat anti-hlgkappa-HRP (Southern Biotechnology, Cat.#2060-05) and goat anti-hIglambda (Southern Biotechnology, Cat.#2070-05) in secondary screening) were added and the plates incubated atroom temperature for 1 hour. After incubation, the plates were washedthree times with washing buffer and 100 μL/well of TMB (BioFX Lab. Cat.#TMSK-0100-01) was added and the plates allowed to develop for about 10minutes. At this time, 50 μL/well stop solution (TMB Stop Solution(BioFX Lab. Cat. #STPR-0100-01) were added and the plates read on anELISA plate reader at wavelength 450 nm. The results presented in Table10 demonstrate that several of the anti-MCP-1 antibodies cross-reactedwith related chemokines.

TABLE 10 rmJE/MCP-1 rat MCP-1 rhMCP-2 rhMCP-3 rhMCP-4 mAb 2 μg/mL 1μg/mL 2 μg/mL 2 μg/mL 2 μg/mL 1.1.1 0.045 0.051 0.051 0.064 0.052 1.2.10.041 0.044 0.056 0.048 0.055 1.3.1 0.046 0.048 0.065 0.052 0.048 1.4.10.042 0.05 0.046 0.049 0.045 1.5.1 0.043 0.045 0.047 0.069 0.05 1.6.10.042 0.062 0.042 0.046 0.044 1.7.1 0.041 0.042 0.044 0.053 0.041 1.8.10.045 0.049 0.048 0.054 0.046 1.9.1 0.053 0.065 0.04 0.044 0.042 1.10.10.041 0.059 0.04 0.047 0.052 1.11.1 0.041 0.052 0.041 0.043 0.043 1.12.10.042 0.062 0.042 0.046 0.044 1.13.1 0.043 0.06 0.046 0.047 0.045 1.14.10.042 0.062 0.042 0.046 0.044 1.18.1 0.044 0.058 0.04 0.045 0.045 2.3.10.054 0.058 0.052 0.059 0.064 2.4.1 0.129 0.077 0.045 0.066 0.06 3.4.10.044 0.053 0.042 0.05 0.047 3.5.1 0.042 0.053 0.042 0.045 0.044 3.6.10.047 0.046 0.052 0.045 0.048 3.7.1 0.046 0.048 0.043 0.048 0.048 3.80.042 0.062 0.042 0.046 0.044 3.10.1 0.054 0.045 0.845 0.167 0.0423.11.1 0.063 0.057 0.336 1.317 0.981 3.14.1 0.044 0.046 0.045 0.05 0.0453.15.1 0.041 0.05 0.043 0.046 0.051 3.16.1 0.042 0.046 0.049 0.043 0.0434.5.1 0.049 0.055 0.042 0.046 0.046 4.6.1 0.049 0.05 0.047 0.05 0.0474.7.1 0.042 0.062 0.042 0.046 0.044 4.8.1 0.042 0.091 0.041 0.043 0.0394.9.1 0.05 0.05 0.046 0.049 0.05 5.1 0.044 0.054 0.051 0.05 0.043 5.2.10.04 0.054 0.041 0.048 0.041 5.3.1 0.05 0.047 0.043 0.045 0.043 3.20.059 0.07 0.535 0.449 0.041 (neat) nc 0.042 0.134 0.045 0.084 0.074 pc0.263 ND ND 1.084 0.215 Positive control hGRO/ hMCP- MGSA hMIP-1-alphahRANTES hEotaxin 1(MCAF) mAb 1 μg/mL 1 μg/mL 1 μg/mL 1 μg/mL 2 μg/mL1.1.1 0.047 0.044 0.044 0.042 0.944 1.2.1 0.044 0.04 0.04 0.044 1.1591.3.1 0.051 0.049 0.049 0.046 1.158 1.4.1 0.044 0.041 0.046 0.043 0.7381.5.1 0.048 0.041 0.049 0.043 1.178 1.6.1 0.046 0.046 0.046 0.042 0.3751.7.1 0.041 0.04 0.039 0.04 1.17 1.8.1 0.06 0.045 0.045 0.047 1.1591.9.1 0.043 0.044 0.042 0.042 0.446 1.10.1 0.043 0.043 0.042 0.05 1.2591.11.1 0.042 0.042 0.042 0.049 1.336 1.12.1 0.046 0.046 0.046 0.0440.933 1.13.1 0.046 0.042 0.046 0.044 1.16 1.14.1 0.046 0.046 0.046 0.0421.129 1.18.1 0.049 0.043 0.04 0.043 1.228 2.3.1 0.062 0.067 0.055 0.0450.087 2.4.1 0.048 0.061 0.046 0.084 0.462 3.4.1 0.065 0.055 0.046 0.0481.153 3.5.1 0.048 0.047 0.044 0.043 0.194 3.6.1 0.047 0.047 0.043 0.0430.342 3.7.1 0.045 0.049 0.067 0.043 1.276 3.8 0.046 0.046 0.046 0.0420.275 3.10.1 0.042 0.043 0.04 0.306 0.71 3.11.1 0.054 0.053 0.064 0.3390.803 3.14.1 0.046 0.046 0.045 0.043 0.549 3.15.1 0.044 0.045 0.0490.045 0.948 3.16.1 0.043 0.043 0.042 0.043 0.633 4.5.1 0.045 0.046 0.0490.041 0.957 4.6.1 0.046 0.055 0.053 0.049 0.686 4.7.1 0.046 0.046 0.0460.042 0.744 4.8.1 0.042 0.041 0.044 0.043 1.136 4.9.1 0.043 0.049 0.0570.045 0.822 5.1 0.044 0.043 0.043 0.042 0.521 5.2.1 0.045 0.043 0.2620.043 0.663 5.3.1 0.045 0.042 0.045 0.042 0.272 3.2 0.042 0.041 0.0430.194 0.235 (neat) nc 0.357 0.065 0.072 0.063 0.042 pc 1.075 0.794 1.2190.221 0.281 Coat: Ag @ 2 μg/mL or 1 μg/mL; O/N Ab: MCP-1 purified clones1:50 pc: 1 μg/mL; nc: D39.2 IL8 @1 μg/mL Detect samples with gxhG-Fc HRP1:2K; controls with mix xmIgG1, 2a, 2b, 3 1:1K

To determine whether anti-MCP-1 antibody 3.11.2 could block the functionof other MCP family members, migration assays as described above wereperformed. First, the ability of THP-1 monocytes to migrate in responseto MCP-1, MCP-2, MCP-3, and MCP-4 was determined. MCP-1, -2 and -3effectively induced migration of THP-1 cells, but MCP-4 was not activein this assay (see FIG. 1). When antibody 3.11.2 was added to the bottomside of the well at varying concentrations, the ability of the THP-1cells to migrate in response to MCP-2 and MCP-3 was inhibited in a dosedependent manner (FIGS. 2 and 3).

Example 3 Epitope Mapping of MCP-1

Monocyte chemo-attractant protein-1 (MCP-1) is a member of the betachemokine family that acts through a specific seven-transmembranereceptor to recruit monocytes, basophils, and T lymphocytes to the siteof inflammation. The antigen, a 76-amino-acid residue is nonglycosylatedand has a predicted molecular mass of 8.7 kD. Human MCP-1, expressed inE. coli, was purchased from R&D #279MC/CF. Monkey MCP was expressed in293F cells, and three monkey MCP-1 variants were used to analyze howdefined amino-acid replacements affect binding affinity for eachindividual mAb.

Sequence analysis showed that the antibodies fell into five classes. Thelargest class included 28 antibodies highly related by their use ofVH1-24, of which, 24 also use Vk gene B3. A class comprised of threeantibodies use the VH6-1 gene, two of which use Vk B3. Three otherclasses are represented by one antibody each, using VH1-2, VH3-33 andVH4-31, of which two of these mAbs use the Vk08 gene. It should be notedthat antibody names beginning with 1, 2, 3, or 4 represent differenthybridoma fusions from independent cohorts of XenoMouse® mice.Therefore, these monoclonal antibodies arose from independent lineagesof B cells maturing during independent primary and secondary immuneresponses in XenoMouse® mice. Because of their independence, thesimilarity in nucleotide and amino acid sequence of the antibody VH andVk genes likely represents a convergent evolution and selection for asimilar variable region structure that can bind to and potentlyneutralize MCP-1 (see Table 11).

TABLE 11 Iso- Samples type VH DH JH VK JK Epitope 1.1.1 γ2/κ VH1-24D3-3(17) JH4b VK-B3 JK1 Conf. 1.2.1 γ2/κ VH1-24 D3-3(17) JH4b VK-L5 JK1Linear 1.3.1 γ2/κ VH1-24 D3-3(15) JH4b VK-B3 JK1 Conf. 1.4.1 γ2/κ VH6-1D1-26 JH4b VK-A2 JH4 linear 1.5.1 γ2/κ VH1-24 D3-3(17) JH4b VK-B3 JK1Linear 1.6.1 γ2/κ VH1-24 D1-26(18) JH3b VK-A10 JK4 Conf. 1.7.1 γ2/κVH1-24 D3-3(17) JH4b VK-B3 JK1 Conf. 1.8.1 γ2/κ VH1-24 D3-3(17) JH4bVK-B3 JK1 Linear 1.9.1 γ2/κ VH1-24 D5-12(13) JH4b VK-B3 JK1 no binding1.10.1 γ2/κ VH1-24 D3-3(17) JH4b VK-B3 JK1 Linear 1.11.1 γ2/κ VH1-24D3-3 JH4B VK-B3 JK1 Linear 1.12.1 γ2/κ VH1-24 D3-3(16) JH4b VK-B3 JK1Conf. 1.13.1 γ2/κ VH1-24 D3-3(17) JH4b VK-B3 JK1 Linear 1.14.1 γ2/κVH6-1 D1-26 JH6b VK-B3 JK1 Linear 1.18.1 γ2/κ VH1-24 D3-3(15) JH4b VK-B3JK4 Linear 2.3.1 γ4/κ VH1-24 D3-3(16) JH4b VK-B3 JK2 no binding 3.2 γ2/κVH1-24 D3-3(17) JH4b VK-L16 JK4 Conf. 2.4.1 γ4/κ VH1-2 D6-13(15) JH4bVK-08 JK5 no binding 3.4.1 γ2/κ VH1-24 D3-3(16) JH4b VK-B3 JK1 Linear3.5.1 γ4/κ VH1-24 D3-3(17) JH4b VK-B3 JK1 no binding 3.6.1 γ4/κ VH1-24D3-3(17) JH4b VK-B3 JK1 no binding 3.7.1 γ2/κ VH1-24 D3-3(16) JH4b VK-B3JK1 Conf. 3.8 γ4/κ VH1-24 D3-3 JH4B VK-B3 JK1 no binding 3.10.1 γ4/κVH1-24 D3-9(12) JH6b VK-A30 JK3 Conf. 3.11.1 γ4/κ VH4-31 D2-21(10) JH3bVK-08 JK2 Conf. 3.14.1 γ4/κ VH6-1 D1-26 JH6B VK-B3 JK1 Conf. 3.15.1 γ4/κVH1-24 D5-12(13) JH4b VK-B3 JK1 Linear 3.16.1 γ4/κ VH1-24 D3-3(17) JH4bVK-B3 JK1 Conf. 4.5.1 γ2/κ VH1-24 D3-3(16) JH4b VK-B3 JK1 Conf. 4.6.1γ2/κ VH1-24 D3-3 JH3B VK-B3 JK1 ND 4.7.1 γ2/κ VH1-24 D3-3(16) JH4b VK-B3JK1 Conf. 4.8.1 γ2/κ VH1-24 D3-3 JH4b VK-B3 JK1 Conf. 4.9.1 γ2/κ ND NDND ND ND Conf. 5.1 γ2/λ VH3-33 D6-6(15) JH6B V1-22 JK2 ND 5.3.1 γ2/κVH1-24 D5-12(13) JH4b VK-B3 JK1 no binding Conf. = conformational ND =Not Done No binding = No binding on western blot.

Whether each antibody bound to a linear or conformational epitope wasdetermined by Western blot analysis. To determine whether disruption ofthe intramolecular bonds by a reducing agent changed the reactivity ofselected anti-MCP-1 antibodies, purified MCP-1 was loaded on SDS/PAGE(4–20% gel) under non-reducing (NR) or reducing (R) conditions. SDS/PAGEwas performed by the method of Laemmli, using a mini-gel system.Separated proteins were transferred onto nitrocellulose membrane.Membranes were blocked using PBS containing 5% (w/v) non-fat dried milkfor at least 1 hour before developing, and probed for 1 hour with eachantibody. Anti-MCP-1 antibodies were detected using HRP-conjugated goatanti-human immunoglobulins (1:8,000 dilution; Sigma Catalog No. A-8667).Membranes were developed by using enhanced Chemiluminescence (ECL®;Amersham Bioscience) according to the manufacturer's instructions.

Antibody-MCP-1 complexes were analyzed by three methods: (1) SurfaceEnhanced Laser Desorption Ionization (SELDI) (Protein chip technology)for linear and conformational epitopes; (2) Site Directed Mutagensis forlinear and conformational epitopes; and (3) SPOTs Peptide Array forlinear epitopes. SELDI is a recently developed method for accurate,rapid and sensitive determination of the molecular weights of peptidesand proteins. Linear and conformational epitopes were mapped based onthe mass of the bound fragment to immobilized antibody by SELDI proteinchip technology. Mapping of linear epitopes by SELDI was carried out inthree steps. In the first step, MCP-1 was digested by highly specificproteolytic enzymes to generate sets of peptide fragments. In the secondstep, peptide fragments containing the linear epitopes were selected bytheir specific binding to the immobilized antibody on the protein chip.In this step, peptides that contain the epitope form complexes with theantibody, while other peptides that do not bind the antibody wereremoved by stringency wash. In the final step, the identity of theantibody-binding peptide was determined by its molecular weight by SELDIand the known digestion sites of the specific protease.

Antibodies 1.4.1, 1.8.1, 1.14.1, 1.18.1 reacted equally with native anddenatured MCP-1 on the Western blot, indicating that these have a linearepitope. Their epitope was mapped by SELDI. The experiments were carriedout by carboxymethylation of MCP-1 antigen to prevent the formation ofdisulfide bonds between cysteine residues in the protein. MethylatedMCP-1 was digested with Glu-C, an endoproteinase that specificallycleaves peptide bonds on the carboxy-terminal side of glutamic acid (E)residues. mAbs were covalently coupled to the Protein chip array, PS20.The chip surface was blocked with 1M ethanolamine and washed with PBS,0.5% Triton. Glu-C fragments of methylated MCP-1 antigen were bound tothe immobilized antibody. Unbound fragments were washed off withdetergent (PBS, 0.1% Tween). Bound Glu-C fragments (epitope) wereanalyzed and identified by SELDI based on their mass. Table 12summarizes the expected mass of each peptide generated from completedigest of methylated MCP-1 with Glu-C. MCP-1 was completely digestedinto three fragments. The theoretical pI was: 9.39/Mw (average mass):8685.03/Mw (monoisotopic mass): 8679.44. After the wash, the fragmentwith the mass 4635, corresponding to the residues 1–39, remained boundto the antibody, indicating that the epitope of all these antibodieslies in the first 39 residues as same pattern was seen with each ofthese antibodies.

TABLE 12 Position in SEQ ID Artif. Mass NO: 149 #MC modification(s)Peptide sequence 4458.2591  1–39 0 Cys_CM: 11, 12, 36QPDAINAPVTCCYNFTNRKI 4632.2755 SVQRLASYRRITSSKCPKE 3041.4819 51–76 0Cys_CM: 52 ICADPKQKWVQDSMDHLDKQ 3099.4873 TQTPKT 1218.7456 40–50 0AVIFKTIVAKE

The SELDI approach was also used to map conformational epitopes. In thiscase, the protein A covalently bound to PS2 Protein chip arrays(Ciphergen Biosystems) was used to capture the mAbs, and subsequentlyincubated with MCP-1. After removal of unbound material, the complexeswere digested with high concentration of specific proteases. MCP-1antibodies (1.7.2, 3.11.2 and 3.7.2) do not bind to the reduced,denatured antigen on Western blots, indicating that the epitope islikely to be conformational. Antibodies 1.7.2 and 3.7.2 were firstcovalently coupled to the PS20 chip. Native MCP-1 was bound to theantibody and then digested with an endoproteinase (Lys-C in oneexperiment and Asp-N in the other). Unbound fragments were washed offwith PBS+, 0.2% Triton followed with PBS and HPLC water wash. Theepitope was determined by SELDI and identified by the mass of thefragment. Both these antibodies 1.7.2 and 3.7.2 had a fragment of mass5712 corresponding to the residues 3–53 (Table 13; Theoretical pI:9.39/Mw (average mass): 8685.03/Mw (monoisotopic mass): 8679.44) boundto it after the wash, indicating that the epitope lies in the 3 to 53amino acid residues of the native MCP-1 antigen.

TABLE 13 Position in SEQ Mass ID NO: 149 #MC Peptide sequence 5720.0059 3–53 0 DAINAPVTCCYNFTNRKISV QRLASYRRITSSKCPKEAVI FKTIVAKEICA 1046.547668–76 0 DKQTQTPKT 1028.5523 54–61 0 DPKQKWVQ

For mapping the epitope of the antibody 3.11.2, the size of the bindingdomain was minimized by using a different protease. Protein A(Calbiochem, 539202) was immobilized covalently to a PS20 chip. Residualbinding sites were blocked with ethanolamine, pH 8.0. Antibody 3.11.2was bound to protein A. The chip was washed with PBS and then with 50 mMHepes, pH 7.5. MCP-1 antigen was bound to the antibody. Unbound antigenwas removed by washing with 0.1% Tween in PBS, followed by 50 mM Hepes,pH 7.5, and 100 mM ammonium bicarbonate. One chip digestion of MCP-1 wascarried out with the endoproteinase, Lys-C. The chip was washed with0.1% Triton in PBS to remove the unbound fragments. The bound fragmentwas analyzed based on its mass on SELDI. Only one peak of mass 1861.8was bound to the antibody, representing a 15-amino-acid sequence,located at residues 20 to 35 (Table 14; Theoretical pI: 9.39/Mw (averagemass): 8685.03/Mw (monoisotopic mass): 8679.44) of MCP-1, with the massof 1865 and the sequence ISVQRLASYRRITSSK (Position 20–35 of SEQ ID NO.:149) was identified as the most tightly bound fragment.

TABLE 14 Position in SEQ Mass ID NO: 149 #MC Peptide sequence 2155.0059 1–19 0 QPDAINAPVTCCYNFTNRK 1865.0715 20–35 0 ISVQRLASYRRITSSK 1373.615459–69 0 WVQDSMDHLDK 775.3654 50–56 0 EICADPK 706.4134 39–44 0 EAVIFK702.3781 70–75 0 QTQTPK 531.3500 45–49 0 TIVAK

Mutagenesis of MCP-1. It was previously shown that two clusters ofprimarily basic residues (R24, K35, K38, K49, and Y13) appear to makethe largest contributions to the interaction between MCP-1 and itsreceptor (Hemmerich et al., (1999) Biochemistry 38, 13013–13025).Binding data reveled that the N-terminal residues contribute little tobinding activity and that two important residues are important forsignaling activity of the MCP-1: K35 and R24. K35 is the mostfunctionally important residue, because K35A mutation has a significanteffect on binding and activity, as well as alanine mutants of R24(Hemmerich et al., (1999) Biochemistry 38, 13013–13025). Arg24 isconserved across different species of MCP-1 as well as in human MCP-2-4,but varies widely in other CC chemokines and therefore maybe involved inreceptor specificity. To identify individual residues within the first39 residues of MCP-1, representing the Glu-C digest, that were importantfor antibody binding, three MCP-1 mutants were generated: the threebasic residues, R24, K35, and K38, were mutated by site-directedmutagenesis and mutant protein was further analyzed for binding to all36 neutralizing antibodies by ELISA. Arg24 was mutated to alanine (R24A)and glutamic acid (R24E). Lys35 and K38 were mutated to alanine (K35A,K38A respectively). All mutations were introduced in Monkey MCP-1background. The monkey MCP-1 construct was generated recovered byperforming RT-PCR on RNA isolated from monkey peripheral bloodlymphocytes (cynomologus MCP-1 PCR3.1 bidirectional). Protein sequencealignment between human and Monkey MCP-1 reveled 99% homology with twoamino-acids changes at the C-terminal (positions 71 and 76). TheC-terminal residues 59–76 are not involved in interaction with thereceptor and did not affect the binding of all 36 antibodies.

ELISA assays were performed using supernatant from 293 cells transfectedwith different MCP-1 mutated constructs. ELISA plates were coated withanti-human MCP-1 goat IgG Polyclonal antibody (R&D catalog No. AF279NA)diluted to 1 μg/mL in ELISA plate coating buffer. Expression of mutantMCP-1 constructs in 293 cells was confirmed by detection withbiotinylated goat anti-human MCP-1 (R&D catalog No. BAF279) followed bystreptavidin HRP. Binding of mutant MCP-1 to MCP-1 antibodies wasdetected with HRP conjugated goat anti-human IgG (Fc specific, CaltagCatalog No. H10507). ELISA results have shown that changing K38 did nothave any effect of binding activity of all 36 antibodies. Binding of allantibodies to R24E and R24A MCP-1 mutant antigen was completelyabolished (see Table 15). However, the K35A mutation inhibited thebinding of only six antibodies (1.6.1, 1.9.1, 3.6.1, 3.10.1). All ofthese antibodies appear to have a conformational epitope, binding towhich is affected by mutation of either Arg24 or Lys35. These datasuggest that these four antibodies recognize a conformational epitopedifferent, but overlapping with, the other antibodies.

TABLE 15 Glu-C mAb Epitope digest Lys-C Asp-N digest Peptide ResiduesR24A/E K35A 1.1.1 Conf. ND ND ND ND ND Inhibition Inhibition 1.2.1Linear ND ND ND 7_11 21–25 Inhibition No Inhibition 1.3.1 Conf. ND ND NDND ND Inhibiton No Inhibition 1.4.1 Linear 1_39 ND ND 7_11 21–25Inhibition No Inhibition 1.5.1 Linear ND ND ND 7_11 21–25 Inhibition NoInhibition 1.6.1 Conf. ND ND ND ND ND Inhibition Inhibition 1.7.1 Conf.ND ND 3–53/5712 ND ND Inhibition No Inhibition 1.8.1 Linear 1_39 ND ND7_11 21–25 Inhibition No Inhibition 1.9.1 no binding ND ND ND ND NDInhibition Inhibition 1.10.1 Linear ND ND ND 7_11 21–25 Inhibition NoInhibition 1.11.1 Linear ND ND ND ND ND Inhibition No Inhibition 1.12.1Conf. ND ND ND ND ND Inhibition No Inhibition 1.13.1 Linear ND ND ND7_11 21–25 Inhibition No Inhibition 1.14.1 Linear 1_39 ND ND 7_11 21–25Inhibition No Inhibition 1.18.1 Linear 1_39 ND ND 7_11 21–25 InhibitionNo Inhibition 2.3.1 no binding ND ND ND ND ND Inhibition No Inhibition3.2 Conf. ND ND ND ND ND Inhibition No Inhibition 2.4.1 no binding ND NDND ND ND Inhibition No Inhibition 3.4.1 Linear ND ND ND ND ND InhibitionNo Inhibition 3.5.1 no binding ND ND ND ND ND Inhibition No Inhibition3.6.1 no binding ND ND ND ND ND Inhibition Inhibition 3.7.1 Conf. ND ND3–53/5712 ND ND Inhibition No Inhibition 3.8 no binding ND ND ND ND NDInhibition Inhibition 3.10.1 Conf. ND ND ND ND ND Inhibition Inhibition3.11.1 Conf. ND 20–35(1864) ND ND ND Inhibition No Inhibition 3.14.1Conf. ND ND ND ND ND Inhibition No Inhibition 3.15.1 Linear ND ND ND7_11 21–25 Inhibition No Inhibition 3.16.1 Conf. ND ND ND ND NDInhibition No Inhibition 4.5.1 Conf. ND ND ND ND ND Inhibition NoInhibition 4.6.1 ND ND ND ND ND ND Inhibition No Inhibition 4.7.1 Conf.ND ND ND ND ND Inhibition No Inhibition 4.8.1 Conf. ND ND ND ND NDInhibition No Inhibition 5.1 ND ND ND ND ND ND Inhibition No Inhibition5.3.1 no binding ND ND ND ND ND Inhibition No Inhibition ND = Not DoneNo binding = No binding on Western blot.

For those antibodies binding to a linear epitope, their binding to apeptide epitope was studied in detail using the SPOTs technology. SPOTsis a technology that allows the solid-phase synthesis of hundreds ofpeptides in a format suitable for the systematic analysis of antibodyepitopes. The system is simple, extremely rapid and economic in its useof reagents. A custom-made peptide array was obtained from Sigma-Genosys(The Woodlands, Tex.). A series of 32, 13-mer peptides were synthesizedspanning residues 1–76 of the MCP-1 sequence. Each consecutive peptidewas offset by two amino acids from the previous one, yielding a nested,overlapping library. The membrane carrying the 32 peptides was probedwith eight MCP-1 antibodies (1 μg/mL), detected with HRP-conjugatedsecondary antibody and followed by enhanced chemiluminescence (ECL).Reaction was observed with five consecutive peptide spots (7 to 11)corresponding to amino acids 21 to 25 of MCP-1. From these results, itappears that the core of the epitope for all of the tested MCP-1antibodies binding to a linear epitope is SVQRL (21–25). The MCP-1sequence is:

-   -   QPDAINAPVTCCYNFTNRKISVQRLASYRRITSSKCPKEAVIFKTIVAKEICADPK        QKWVQDSMDHLDKQTQTPKT (SEQ ID NO: 149)

Eight antibodies, which recognized a linear epitope, reacted with thesame SPOTs: 1.2.1, 1.4.1, 1.5.1, 1.8.1, 1.10.1, 1.13.1, 1.14.1, and1.18.1.

Example 4 Affinity Determination of Cross-Reacting Antibodies byHigh-Resolution Biacore Analysis

The interaction analysis was performed at 25° C. using two CM5 chipsdocked in Biacore 2000 optical biosensors. Individual flow cells on eachchip were activated with a 7-minute injection of NHS/EDC, carbohydrazidewas coupled through the NHS ester using a 7-minute injection, and theresidual activated groups were blocked with a 7-minute injection ofethanolamine. The monosaccharide residues of mAb 3.11.2, diluted 1/50,were oxidized using 1 mM sodium metaperiodate in 100 mM sodium acetate,pH 5.5 at 4° C. for 30 minutes. The oxidized antibody was desalted into10 mM sodium acetate, pH 5.0, to couple the antibody to thecarbohydrazide-modified surface. A surface density of 250 RU mAb 3.11.2was used to measure the reported interactions of MCP-1 and MCP-4, whilea surface of 110 RU was used to measure the interactions of antigensMCP-2 and MCP-3 with mAb 3.11.2. The mAb surfaces were stabilized byreducing the hydrazone bond with 0.1 M sodium cyanoborohydride. Theantigen/antibody interaction was tested by injecting duplicate antigensamples diluted in running buffer (10 mM HEPES, 150 mM NaCl, 0.005%surfactant, 200 μg/mL BSA, pH 7.4), in a 300-fold concentration range.The surfaces were regenerated with a 12-second pulse of 15 mM H₃PO₄.

To determine the kinetics of each interaction, the data sets were fitglobally to a 1:1 interaction model that included a parameter for masstransport. The estimated rate constants and the calculated affinities ofinteraction for antibody 3.11.2 are reported in Table 16. The data forall the other antibodies are presented in Table 8.

TABLE 16 Ag k_(a) (M⁻¹ s⁻¹) k_(d) (s⁻¹) K_(D) (pM) MCP-1 3.0 × 10⁸ 1.0 ×10⁻³ 3.3 MCP-2 2.6 × 10⁸ 1.2 × 10⁻² 46 MCP-3 1.5 × 10⁸ 7.4 × 10⁻³ 49MCP-4 1.5 × 10⁸ 5.5 × 10⁻⁴ 3.7

Example 5 Prevention of Angiogenesis with Antibodies to MCP-1

Angiogenesis was induced in a mouse model by admixing Matrigel withhuman bFGF (10 ng/mL), human VEGF165 (100 ng/mL) and 10 g/mL heparin orMCP-1 (250 ng/mL) and MCP-3 (100 ng/mL). About 0.5 mL of the suspensionwas subcutaneously injected into the right flank of 6–8 week-old,athymic, female, nude mice. Five mice were used for each dose of MCP-1and MCP-3. In addition, as a negative control, Matrigel alone (no growthfactors) was included. The Matrigel implants solidified in situ and wereleft undisturbed for 7 days. At the end of 7 days, the mice wereanesthetized, and the Matrigel plugs were removed carefully usingmicrosurgical instruments. Gels were photographed undertransillumination. One part of the plugs was processed for paraffinembedded sectioning. Sections were cut at two different levels andstained with H/E. Another part of the gel was snap frozen in liquidnitrogen and subjected to immunocytochemical staining with ratmonoclonal antibody directed against mouse CD31 antigen conjugated withphycoerythrin. H+E stained slides were elevated for the formation of thedistinct, endothelial lined vessels. Anti-CD31-PE stained slides wereobserved under Fluorescence microscope (red filter) attached to a SpotCamera. Images were captured digitally using Metamorph software program.Microvessel density was determined by the method published by Wild etal. (2000).

Both MCP-1 and MCP-3 were found to show equivalent angiogenesis as thewell-characterized angiogenic factors VEGF and bFGF. In addition,angiogenesis induced by MCP-1 or MCP-3 in animals, and by inference inhuman tumors or diseased tissue, can be prevented by treating withantibodies to MCP-1 or an antibody such as 3.11.2, which neutralizes theactivity of all MCP family members. Accordingly, one would inject theanti-MCP antibodies into animals at different doses ranging fromapproximately 0.1 to 0.5 mg per animal to obtain a dose-responserelationship for treatment.

Example 6 MCP-1 Production by Tumor Cells

To determine whether tumor cells produced MCP-1 in cell culture, a panelof cell lines was examined for their ability to secrete MCP-1 into theculture medium. Cells were cultured in Dulbecco's Modified Eagles Medium(DMEM) containing 10% fetal bovine serum or an equivalent untilconfluent. The supernatant was removed and an aliquot tested forreactivity to MCP-1 using a commercially available ELISA kit from R & DSciences. Table 17 shows a series of cancer cell lines thatconstitutively secrete MCP-1 and their respective MCP-1 levels asdetermined by ELISA.

TABLE 17 Cell Line MCP-1 (pg/mL) 1 Colon Carcinoma COLO-205 <10 2 ColonCarcinoma HCT-15 60 3 Colon Carcinoma HCT-116 122 4 Colon CarcinomaHT-29 102 5 Cervical Cancer HT-3 127 6 Colon Carcinoma SW707 31 7 ColonCarcinoma SW948 13 8 Colon Carcinoma KM-12 6 9 Colon Carcinoma HCC-299839 10 Gastric Carcinoma NCI-N87 37 11 Gastric Carcinoma NCI-SNU-1 4 0 12Gastric Carcinoma NCI-SNU-5 <10 13 CNS Carcinoma SF-268 94 14 CNSCarcinoma SF-295 223 15 CNS Carcinoma SF-593 >2500 16 CNS CarcinomaSNB-19 >2500 17 CNS Carcinoma SNB-75 >2500 18 CNS Carcinoma U251 >250063 CNS XF-498(Curg) >2500 61 Glioblastoma SF-295(Curg) >2500 21Medulloblastoma TE 671 (u) >2500 25 Leukemia SR 25 26 Leukemia A673 >2501 27 Leukemia K562 287 28 Leukemia RPMI-8226 528 29 LeukemiaJurkats 184 30 Leukemia THP-1 113 31 Leukemia HUT 78 35 32 Leukemia JY 033 Leukemia CEM 0 34 Lung Carcinoma MV 522 74 35 Lung adenocarcinomaEKVX >2500 36 Lung adenocarcinoma HOP-62 >2500 37 Lung Carcinoma NSCHOP-92 897 38 Lung Carcinoma NSC NCI-H1299 384 39 Lung Carcinoma NSCNCI-H2126 107 55 Lung adenocarcinoma NCI-H522 0 42 Lung adenocarcinomaNCI-H322M 0 40 IPF Lung fibroblasts A 549 >2501 57 Lung adenocarcinomaNCI-H292 245 43 Lung Carcinoma NSC NCI-H460 118 45 Lung Squamous NSCSkmes-1 410 44 Lung Carcinoma Small Cell SHP-77 1663 58 Lung CarcinomaSmall Cell NCI-H510A >2500 56 Lung Carcinoma Small Cell NCI-H69 53Mammary Gland Carcinoma HCC-2218 129 54 Mammary Gland Carcinoma HCC-1954113 46 Mammary Gland Carcinoma ZR-75-30 357 47 Mammary Gland CarcinomaMCF-7 0 48 Mammary Gland Carcinoma MDA-MB-453 40 49 Mammary GlandCarcinoma MDA-MB-231 >2501 50 Mammary Gland Carcinoma MDA-MB-468 9 51Mammary Gland Carcinoma NCI/ADR 0 52 Mammary Gland Carcinoma T47D 61 22Mammary Gland Carcinoma SK-BR-3 475 20 Mammary Gland Carcinoma Hs605T >2500 53 Melanoma A431 56 54 Melanoma LOX IMVI 105 55 Melanoma M14786 56 Melanoma RPMI 7591 >2501 57 Melanoma SK-MEL-28 29 58 MelanomaUACC-62 119 59 Melanoma UACC-257 265 41 Melanoma Hs 936.T 15 24 MelanomaSK-mel-5 38 25 Melanoma Hs 940.T >2500 26 Melanoma A375 136 6 MelanomaWM.266.4 >2500 27 Pancreatic Carcinoma HPAC 73 29 Pancreatic CarcinomaHPAF II 47 41 Pancreatic Carcinoma CAPAN-1 >2500 60 Pancreatic CarcinomaPanc-1 >2500 30 Ovarian Carcinoma ES2 322 31 Ovarian Carcinoma IGROV1199 32 Ovarian Carcinoma MDAH2774 314 33 Ovarian Carcinoma SK-OV-3 86 34Ovarian Carcinoma OVCAR-3 126 36 Ovarian Carcinoma OVCAR-5 336 37Ovarian Carcinoma OVCAR-8 36 38 Prostate Carcinoma 22Rv1 55 39 ProstateCarcinoma LNCaP >2500 40 Prostate Carcinoma DU150 >2500 42 ProstateCarcinoma PC-3 163 28 Prostate Carcinoma DU145 68 43 Renal CarcinomaA498 >2500 44 Renal Carcinoma 786-0(35h) >2500 45 Renal CarcinomaSK-RC-01 >2500 46 Renal Carcinoma SK-RC-10 >2500 47 Renal CarcinomaCaki-1 115 48 Renal Carcinoma Caki-2 >2500 49 Renal CarcinomaRXF-393 >2500 50 Renal Carcinoma SK-RC-52 >2500 51 Renal CarcinomaSN12C >2500 52 Renal Carcinoma TK-10 533 62 Renal Carcinoma 769-P 512 23Liver Carcinoma C3A 0 59 Liver Carcinoma HepG2 >2500 19 Cervical CancerEpidermoid MS 751 >2500 35 Cervical Cancer Hela >2501 Cervical C-33A 201 Cervical Ca Ski 32 2 Cervical ME-180 54 3 Uterus KLE >2500 4 UterusRL95-2 28 5 Uterus HEC-1-A 47 MCP-1

Example 7 Effect of Anti-MCP-1 Antibodies in Mouse Tumor Model

To evaluate the effect of anti-MCP-1 antibodies on the growth of asubcutaneous tumor, exponentially growing Panc-1 cells were harvestedand resuspended in 0.2 ml of Hank's Balanced Salt solution (HBSS).Tumors were produced following the injected of 5×10⁶ Panc-1 cellsadmixed with Growth factor reduced Matrigel into the flanks of femaleBALB/c nude mice. Beginning on the day of implantation, animals weretreated with 5 mg of anti-MCP-1 antibody 1.7.3, and antibody PK, whichwas directed to KLH or PBS at the times indicated on the graph. Tumorgrowth was monitored weekly and the results presented as mean±SD (FIG.4). The difference between the control and treated animals wasstatistically significant when compared using the student T test(P<0.002). Accordingly, anti-MCP-1 antibodies provide an effectivetreatment for reducing tumor growth in vivo.

Example 8 Software-Assisted Analysis of MCP-1 Antibodies

The above-described calcium flux, chemotaxis and affinity data for theMCP-1 antibodies were analyzed using Guided Analytic software availablefrom Spotfire, Inc., Somerville, Mass. The results are shown in FIGS. 5and 6.

Example 9 Structural Analysis of Anti-MCP-1 Antibodies

The variable heavy chains and the variable light chains for theantibodies shown in Table 1 were sequenced to determine their DNAsequences. The complete sequence information for all anti-MCP-1antibodies are shown in the sequence listing with nucleotide and aminoacid sequences for each gamma and kappa chain combination.

The variable heavy sequences were analyzed to determine the VH family,the D-region sequence and the J-region sequence. The sequences were thentranslated to determine the primary amino acid sequence and compared tothe germline VH, D and J-region sequences to assess somatichypermutations. FIG. 7 shows a Clustal W comparison of anti-MCP-1sequences using VH1-24, indicating the CD, CDR1, CDR2, and CDR3 regions,and the associated dendrogram. FIG. 8 shows a Clustal W comparison ofanti-MCP-1 sequences using VK-B3, indicating the CD, CDR1, CDR2, andCDR3 regions, and the associated dendrogram. FIG. 9 shows a Clustal Wcomparison of anti-MCP-1 sequences using VK-08, indicating the CD, CDR1,CDR2, and CDR3 regions, and the associated dendrogram. FIG. 10 shows aClustal W comparison of anti-MCP-1 sequences using VH6-1, indicating theCD, CDR1, CDR2, and CDR3 regions, and the associated dendrogram.

Example 10 Use of Anti-MCP-1 Antibodies as a Diagnostic Agent

A. Detection of MCP-1 Antigen in a Sample

An Enzyme-Linked Immunosorbent Assay (ELISA) for the detection of MCP-1antigen in a sample is developed. In the assay, wells of a microtiterplate, such as a 96-well microtiter plate or a 384-well microtiterplate, are adsorbed for several hours with a first fully humanmonoclonal antibody directed against the antigen. The immobilizedantibody serves as a capture antibody for any of the antigen that may bepresent in a test sample. The wells are rinsed and treated with ablocking agent such as milk protein or albumin to prevent nonspecificadsorption of the analyte.

Subsequently the wells are treated with a test sample suspected ofcontaining the antigen, or with a solution containing a standard amountof the antigen. Such a sample may be, for example, a serum sample from asubject suspected of having levels of circulating antigen considered tobe diagnostic of pathology.

After rinsing away the test sample or standard, the wells are treatedwith a second fully human monoclonal anti-MCP-1 antibody that is labeledby conjugation with biotin. The labeled anti-MCP-1 antibody serves as adetecting antibody. After rinsing away excess second antibody, the wellsare treated with avidin-conjugated horseradish peroxidase (HRP) and asuitable chromogenic substrate. The concentration of the antigen in thetest samples is determined by comparison with a standard curve developedfrom the standard samples.

This ELISA assay provides a highly specific and very sensitive assay forthe detection of the MCP-1 antigen in a test sample.

B. Determination of MCP-1 Concentration in Patient Samples

A sandwich ELISA is developed to quantify MCP-1 levels in human serum.The two anti-MCP-1 antibodies used in the sandwich ELISA, preferablyrecognize different epitopes on the MCP-1 molecule (data not shown). TheELISA is performed as follows: 50 μl of capture anti-MCP-1 antibody incoating buffer (0.1 M NaHCO₃, pH 9.6) at a concentration of 2 μg/mL iscoated on ELISA plates (Fisher). After incubation at 4° C. overnight,the plates are treated with 200 μl of blocking buffer (0.5% BSA, 0.1%Tween 20, 0.01% Thimerosal in PBS) for 1 hr at 25° C. The plates arewashed (3×) using 0.05% Tween 20 in PBS (washing buffer, WB). Normal orpatient sera (Clinomics, Bioreclaimation) are diluted in blocking buffercontaining 50% human serum. The plates are incubated with serum samplesovernight at 4° C., washed with WB, and then incubated with 100 μl/wellof biotinylated detection anti-MCP-1 antibody for 1 hr at 25° C. Afterwashing, the plates are incubated with HRP-Streptavidin for 15 min,washed as before, and then treated with 100 μl/well ofo-phenylenediamine in H₂O₂ (Sigma developing solution) for colorgeneration. The reaction is stopped with 50 μl/well of H₂SO₄ (2M) andanalyzed using an ELISA plate reader at 492 nm. Concentration of PROantigen in serum samples is calculated by comparison to dilutions ofpurified MCP-1 antigen using a four-parameter curve-fitting program.

C. Staging of Cancer in a Patient

It will be appreciated that based on the results set forth and discussedin Examples 10A–10B, through use of embodiments of the inventiondescribed herein, it is possible to stage a cancer in a subject based onexpression levels of the MCP-1 antigen. For a given type of cancer,samples of blood are taken from subjects diagnosed as being at variousstages in the progression of the disease, and/or at various points inthe therapeutic treatment of the cancer. The concentration of the MCP-1antigen present in the blood samples is determined using a method thatspecifically determines the amount of the antigen that is present. Sucha method includes an ELISA method, such as the method described inExamples 10A–10B. Using a population of samples that providesstatistically significant results for each stage of progression ortherapy, a range of concentrations of the antigen that may be consideredcharacteristic of each stage is designated.

In order to stage the progression of the cancer in a subject understudy, or to characterize the response of the subject to a course oftherapy, a sample of blood is taken from the subject and theconcentration of the MCP-1 antigen present in the sample is determined.The concentration so obtained is used to identify in which range ofconcentrations the value falls. The range so identified correlates witha stage of progression or a stage of therapy identified in the variouspopulations of diagnosed subjects, thereby providing a stage in thesubject under study.

Example 11 Uses of Anti-MCP-1 Antibodies for Tumor Treatment

To determine the in vivo effects of anti-MCP-1 antibody treatment inhuman patients with tumors, such human patients are injected over acertain amount of time with an effective amount of anti-MCP-1 antibody.At periodic times during the treatment, the human patients are monitoredto determine whether their tumors progress, in particular, whether thetumors grow and metastasize.

A tumor patient treated with anti-MCP-1 antibodies has a lower level oftumor growth and metastasis compared to the level of tumor growth andmetastasis of tumors in tumor patients treated with control antibodies.Control antibodies that may be used include antibodies of the sameisotype as the anti-MCP-1 antibodies tested and further, may not havethe ability to bind to MCP-1 tumor antigen.

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. The embodimentsof the invention described herein are not to be limited in scope by theconstruct deposited, since the deposited embodiment is intended as asingle illustration of certain aspects of the invention and anyconstructs that are functionally equivalent are within the scope of thisinvention.

All references cited herein, including patents, patent applications,papers, text books, and the like, and the references cited therein, tothe extent that they are not already, are hereby incorporated herein byreference in their entirety.

The foregoing description and Examples detail certain preferredembodiments of the invention and describes the best mode contemplated bythe inventors. It will be appreciated, however, that no matter howdetailed the foregoing may appear in text, the invention may bepracticed in many ways and the invention should be construed inaccordance with the appended claims and any equivalents thereof.

1. An isolated human monoclonal antibody that binds to MCP-1 andcomprises a heavy chain polypeptide having the sequence of SEQ ID NO.:62.
 2. The antibody of claim 1, further comprising a light chainpolypeptide having the sequence of SEQ ID NO.:64.
 3. An isolatedantibody immobilized on an insoluble matrix, wherein the antibody is theantibody of claim
 1. 4. A method for assaying the level of monocytechemo-attractant protein (MCP-1) in a patient sample, comprising:contacting the anti-MCP-1 antibody of claim 1 with the patient sample;and detecting the level of MCP-1 in the patient sample.
 5. A methodaccording to claim 4, wherein the patient sample is blood.
 6. Acomposition, comprising the antibody of claim 1, and a pharmaceuticallyacceptable carrier.
 7. A method of treating a neoplastic disease,comprising: selecting an animal in need of treatment for a neoplasticdisease; and administering to said animal a therapeutically effectivedose of the antibody of claim
 1. 8. The method of claim 7, wherein saidneoplastic disease is selected form the group consisting of: breastcancer, ovarian cancer, bladder cancer, lung cancer, glioblastoma,stomach cancer, endometrial cancer, kidney cancer, colon cancer,pancreatic cancer, and prostate cancer.
 9. An isolated human monoclonalantibody that binds to the sequence ISVQRLASYRRITSSK (SEQ ID NO.: 150).10. A method of manufacturing the antibody of claim 1, comprising:immunizing a mammal with a synthetic peptide of MCP-1; recovering alymphatic cell that expresses the antibody of claim 1 from the immunizedmammal; and fusing the lymphatic cell with a myeloid-type cell toprepare a hybridoma cell that produces the antibody of claim
 1. 11. Theantibody of claim 1, wherein said antibody is conjugated to atherapeutic agent.
 12. The antibody of claim 11, wherein saidtherapeutic agent is a toxin.
 13. The antibody of claim 12, wherein saidtoxin is an immunotoxin.
 14. The antibody of claim 11, wherein saidtherapeutic agent is a chemotherapeutic agent.
 15. The antibody of claim14, wherein said chemotherapeutic agent is selected from the groupconsisting of taxol, doxorubicin, cis-platinum, and 5-fluorouracil. 16.The antibody of claim 11, wherein said therapeutic agent is a steroid.17. The antibody of claim 11, wherein said therapeutic agent is aradioisotope.
 18. The antibody of claim 17, wherein said radioisotope isselected from the group consisting of ³H, ¹⁴C, ¹⁵N, ³⁵S, ⁹⁰Y, ⁹⁹Tc,¹¹¹In, ¹²⁵In, and ¹³¹I.
 19. The antibody of laim 9, wherein saidantibody is conjugated to a therapeutic agent.
 20. The antibody of claim19, wherein said therapeutic agent is a toxin.
 21. The antibody of claim20, wherein said toxin is an immunotoxin.
 22. The antibody of claim 20,wherein said therapeutic agent is a chemotherapeutic agent.
 23. Theantibody of claim 22, wherein said chemotherapeutic agent is selectedfrom the group consisting of taxol, doxorubicin, cis-platinum, and5-fluorouracil.
 24. The antibody of 22, wherein said therapeutic agentis a steroid.
 25. The antibody of claim 22, wherein said therapeuticagent is a radioisotope.
 26. The antibody of claim 25, wherein saidradioisotope is selected from the group consisting of ³H, ¹⁴C, ¹⁵N, ³⁵S,⁹⁰Y, ⁹⁹Tc, ¹¹¹In, ¹²⁵In, and ¹³¹I.
 27. The antibody of claim 1, whereinsaid antibody neutralizes the activity of MCP-1.
 28. The antibody ofclaim 3, wherein said antibody neutralizes the activity of MCP-1. 29.The antibody of claim 9, wherein said antibody neutralizes the activityof MCP-1.
 30. The antibody of claim 1, wherein said antibody binds toMCP-1 with a dissociation constant (K_(D)) of approximately 3.0 pM. 31.The antibody of claim 30, wherein said dissociation constant is 3.3 pM.32. The antibody of claim 3, wherein said antibody binds to MCP-1 with adissociation constant (K_(D)) of approximately 3.0 pM.
 33. The antibodyof claim 32, wherein said dissociation constant is 3.3 pM.
 34. Theantibody of claim 9, wherein said antibody binds to MCP-1 with adissociation constant (K_(D)) of approximately 3.0 pM.
 35. The antibodyof claim 34, wherein said dissociation constant is 3.3 pM.
 36. Anisolated human monoclonal antigen binding fragment that binds to MCP-1and comprises a heavy chain polypeptide having the sequence of SEQ IDNO.:
 62. 37. The antigen binding fragment of claimn 36, furthercomprising a light chain polypeptide having the sequence of SEQ ID NO.:64.
 38. The antigen binding fragment of claim 36, wherein said bindingfragment is selected from the group consisting of Fab, Fab′, F(ab′)₂,and F_(v).
 39. The antigen binding fragment of claim 36, wherein saidfragment is conjugated to a therapeutic agent.